Indirect Immunohistochemistry Research Articles

The lipoteichoic acid of Lactobacillus plantarum effect on lymphocyte, VEGF-A and TGF-β expression in male rat dental pulp

ObjectiveLipoteichoic acid from Lactobacillus plantarum (L. plantarum) is a significant virulence factor that exacerbates pulp inflammation. Lipoteichoic acid plays a role in modulating the inflammatory to proliferative phase transition is crucial in determining the outcome of pulp healing or necrosis. This study explores the role of L. plantarum on lymphocytes and the expression of transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor A (VEGF-A) in a male rat model of acute dental pulp injury. DesignThe acute dental pulp model was created in the upper molar of Rattus novergicus using a round bur. Then, the dental pulp was exposed to 10 µg/ml of the lipoteichoic acid of L. plantarum and filled with a temporary filling. In the next 24, 48, and 72 h, each animal was decapitated, and the expression of TGF-β1 and VEGF-A in dental pulp was analyzed using indirect immunohistochemistry, while the lymphocytes analyzed using hematoxyline-eosin staining. ResultLipoteichoic acid of L. plantarum induced acute dental pulp by increasing the lymphocyte number after 48 and 72 h of exposure (p < 0.05). While, inhibiting TGF-β1 expression after 48 and 72 h of exposure (p < 0.05), and VEGF-A was inhibiting after 72 h of exposure (p < 0.05). ConclusionExposure to lipoteichoic acid from L. plantarum significantly accelerates the inflammatory response in the dental pulp. However, this accelerated inflammation disrupts the proliferative phase, potentially leading to more extensive damage to the dental pulp.

Neuronal nicotinic acetylcholine receptor antibodies in autoimmune central nervous system disorders.

Neuronal nicotinic acetylcholine receptors (nAChRs) are abundant in the central nervous system (CNS), playing critical roles in brain function. Antigenicity of nAChRs has been well demonstrated with antibodies to ganglionic AChR subtypes (i.e., subunit α3 of α3β4-nAChR) and muscle AChR autoantibodies, thus making nAChRs candidate autoantigens in autoimmune CNS disorders. Antibodies to several membrane receptors, like NMDAR, have been identified in autoimmune encephalitis syndromes (AES), but many AES patients have yet to be unidentified for autoantibodies. This study aimed to develop of a cell-based assay (CBA) that selectively detects potentially pathogenic antibodies to subunits of the major nAChR subtypes (α4β2- and α7-nAChRs) and its use for the identification of such antibodies in "orphan" AES cases. The study involved screening of sera derived from 1752 patients from Greece, Turkey and Italy, who requested testing for AES-associated antibodies, and from 1203 "control" patients with other neuropsychiatric diseases, from the same countries or from Germany. A sensitive live-CBA with α4β2-or α7-nAChR-transfected cells was developed to detect antibodies against extracellular domains of nAChR major subunits. Flow cytometry (FACS) was performed to confirm the CBA findings and indirect immunohistochemistry (IHC) to investigate serum autoantibodies' binding to rat brain tissue. Three patients were found to be positive for serum antibodies against nAChR α4 subunit by CBA and the presence of the specific antibodies was quantitatively confirmed by FACS. We detected specific binding of patient-derived serum anti-nAChR α4 subunit antibodies to rat cerebellum and hippocampus tissue. No serum antibodies bound to the α7-nAChR-transfected or control-transfected cells, and no control serum antibodies bound to the transfected cells. All patients positive for serum anti-nAChRs α4 subunit antibodies were negative for other AES-associated antibodies. All three of the anti-nAChR α4 subunit serum antibody-positive patients fall into the AES spectrum, with one having Rasmussen encephalitis, another autoimmune meningoencephalomyelitis and another being diagnosed with possible autoimmune encephalitis. This study lends credence to the hypothesis that the major nAChR subunits are autoimmune targets in some cases of AES and establishes a sensitive live-CBA for the identification of such patients.

Ex Vivo Analysis of Cell Differentiation, Oxidative Stress, Inflammation, and DNA Damage on Cutaneous Field Cancerization.

Cutaneous field cancerization (CFC) refers to a skin region containing mutated cells' clones, predominantly arising from chronic exposure to ultraviolet radiation (UVR), which exhibits an elevated risk of developing precancerous and neoplastic lesions. Despite extensive research, many molecular aspects of CFC still need to be better understood. In this study, we conducted ex vivo assessment of cell differentiation, oxidative stress, inflammation, and DNA damage in CFC samples. We collected perilesional skin from 41 patients with skin cancer and non-photoexposed skin from 25 healthy control individuals. These biopsies were either paraffin-embedded for indirect immunofluorescence and immunohistochemistry stain or processed for proteins and mRNA extraction from the epidermidis. Our findings indicate a downregulation of p53 expression and an upregulation of Ki67 and p16 in CFC tissues. Additionally, there were alterations in keratinocyte differentiation markers, disrupted cell differentiation, increased expression of iNOS and proinflammatory cytokines IL-6 and IL-8, along with evidence of oxidative DNA damage. Collectively, our results suggest that despite its outwardly normal appearance, CFC tissue shows early signs of DNA damage, an active inflammatory state, oxidative stress, abnormal cell proliferation and differentiation.

Development of monoclonal antibodies against oropouche virus and its applicability to immunohistochemical diagnosis

Orthobunyavirus oropouche ense virus (OROV), the causative agent of Oropouche fever, is widely dispersed in Brazil and South America, causing sporadic outbreaks. Due to the similarity of initial clinical symptoms caused by OROV with other arboviruses found in overlapping geographical areas, differential diagnosis is challenging. As for most neglected tropical diseases, there is a shortage of reagents for diagnosing and studying OROV pathogenesis. We therefore developed and characterized mouse monoclonal antibodies and, one of them recognizes the OROV nucleocapsid in indirect immunofluorescent (IFA) and immunohistochemistry (IHC) assays. Considering that it is the first monoclonal antibody produced for detecting OROV infections, we believe that it will be useful not only for diagnostic purposes but also for performing serological surveys and epidemiological surveillance on the dispersion and prevalence of OROV in Brazil and South America.

Association of the combined parameters including the frequency of primary cilia, PD-L1, Smoothened protein, membranous β-catenin and cytoplasmic β-catenin expression with the outcome of patients with clear cell renal cell carcinoma.

The objective of this study was to investigate the association and combined prognostic significance of the PD-L1, Smoothened protein and β-catenin expressions in patients with clear cell renal cell carcinoma (ccRCC). The PD-L1, Smoothened protein and β-catenin expression were evaluated in 104 ccRCC patients. All studied tumor samples were acquired from nephrectomy specimens of primary tumors and not from biopsies or metastases. An indirect immunohistochemistry using polyclonal rabbit anti-Smoothened antibody, monoclonal mouse anti-human β-catenin-1 antibody, immunohistochemical assay PD-L1 28-8 pharmDx using monoclonal rabbit anti-PD-L1 antibody and anti-VHL (C- terminal) rabbit antibody was used. Immunohistochemistry was scored semiquantitavely. Median overall survival (OS) was significantly better in patients with lower PD-L1 expression (≤5%), Smoothened protein (SMO) expression (<5%) or cytoplasmic β-catenin expression (≤75%) than in patients with higher expressions of these biomarkers (P<0.001, P=0.047, and P<0.001, respectively). Membranous β-catenin showed an opposite effect with its lower expression (≤75%) being associated with longer OS (P=0.020). There was significant association between PD-1 and PD-L1 expression (P=0.007) and significant association of tumor grade (WHO 2016) with membranous β-catenin (P<0.001), cytoplasmic β-catenin (P=0.005), pVHL (P=0.042), PD-L1 (P=0.049) and PD-1 (P=0.028) expression. The present study provides the first data on the potential association and combined prognostic significance of frequency of primary cilia, PD-L1, Smoothened protein and β-catenin expression with the outcome in clear cell renal cell carcinoma.

A multiplex immunoprofiling approach for detecting the co-localization of breast cancer biomarkers using a combination of Alexafluor - Quantum dot conjugates and a panel of chromogenic dyes

There is a plethora of information embedded in a tissue section that the conventional IHC understands only partially. Predictive biomarkers for precision immuno-oncology heavily dependent on the spatial arrangement of cells and the co-expression patterns in the tissue sections. Here we have explored the versatility of indirect multiplex immunofluorescence (mIF) and indirect multiplex immunohistochemistry (mIHC) for the labeling of breast cancer prognostic markers in routinely processed, formalin-fixed paraffin-embedded (FFPE) tissues at high resolution. The multiplex immunohistochemistry protocol utilized sequential staining for the chromogenic immunolabelling of Estrogen Receptor α (ERα) or Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2), and Nucleoside diphosphate kinase 1 (NM23) by multicolor chromogens in different combinations. A feasible workflow for multiplex immunofluorescence was also effectively standardized for ERα, PR, and HER2 using combinations of commercially available Alexa Fluor and Quantum dots semiconductor nanocrystal conjugated secondary antibodies. Multiplex chromogenic immunolabeling revealed differential expression of the markers on the same slide. Kappa statistics revealed perfect agreement with uniplex immunohistochemistry. For multiplex fluorescence approach, surface receptor detection using Quantum dots and Alexa fluor dyes for cytoplasmic or nuclear markers performed well for profiling multiple co-localized biomarkers on a single paraffin tissue section. The technique developed reveals additional information such as co-expression, spatial relationships, and tumor heterogeneity, providing a deeper insight into developing combinatorial therapeutic strategies in clinical care. This high throughput workflow complements the outcomes of traditional IHC while saving tissue, time, labour, and reagents.

LPS-Induced Neuron Cell Apoptosis through TNF-α and Cytochrome c Expression in Dental Pulp.

Inflammation of the dental pulp tissue caused by bacteria, creating an immunology response of death of the dental pulp, is called apoptosis. The Porphyromonas gingivalis that cause apoptosis is lipopolysaccharide (LPS) through toll-like receptor (TLR) via two different mechanisms, intracellular and extracellular pathways. This study analyzed the role of LPS exposure of neuron cells, tumor necrosis factor-α (TNF-α), and cytochrome c (cyt-c) expression in the dental pulp to predict the possible mechanism of apoptosis. The lower tooth of Sprague Dawley rats was opened and exposed to LPS for 48 hours. Then the neuron cell analyzed histopathology using hematoxylin-eosin, whereas the TNF-α and cyt-c expression with indirect immunohistochemistry using a light microscope. The relationship between neuron cells with TNF-α and cyt-c was analyzed using stepwise regression linear analysis. The LPS exposure showed a lower number of neuron cells and had a relationship with TNF-α expression but not with cyt-c, while compared with control, both TNF-α and cyt-c expression were higher in neuron cells. LPS exposure in dental pulp is possible to stimulate the apoptosis process through extracellular pathways marked by higher TNF-α expression.

Viral-induced inflammation can lead to adverse pregnancy outcomes.

Parvoviruses are DNA viruses of small size. There have been a number of reports indicating the possible effects of B19 infections during pregnancy. These effects include spontaneous abortions, stillbirth, fetal damage, and quite often, fetal anemia with hydrops fetalis.

Allelic variants and immunostaining profile in CXCL12/CXCR4 axis: An investigation of association with prognosis in prostate cancer

Prostate cancer (PCa) is the malignant neoplasm that most commonly affects men and is an important cause of death. It can be detected by changes in serum levels of Prostate Specific Antigen (PSA) and digital rectal examination, but often symptoms do not appear until advanced stages and metastases. The C-X-C Motif Chemokine Ligand 12/C-X-C Motif Chemokine Receptor 4 (CXCL12/CXCR4) axis acts in cell migration and may be involved in the metastatic process. In this context, the aim of this study was to evaluate the allelic variants rs1801157 (CXCL12) and rs2228014 (CXCR4) and the immunostaining of CXCR4 protein as candidates for prognostic markers in PCa. Samples (n = 60) were divided according to prognostic parameters (with and without metastasis at diagnosis) in tree groups: better prognosis, worse prognosis with metastasis at diagnosis and worse prognosis without metastasis at diagnosis, and immunostaining was evaluated by indirect immunohistochemistry, considering tumoral and adjacent tissues from the same patient (n = 120). A significant association was found between the C allele of rs2228014 (CXCR4) and the extraprostatic extension. For CXCR4 immunostaining a weak labeling and a cytoplasmic localization predominated, as well as a significant difference between malignant versus adjacent tissue, with higher protein expression in the malignant tissue. A significant association was found between CXCR4 tumor immunostaining with TNM staging (T2b-T2c) and PSA level (> 20 ng/mL). None of the allelic variants affected CXCR4 immunostaining. Prognostic groups did not differ in allelic variant frequency or immunostaining profile. Findings suggest that CXCR4 receptor may be one of the ways to worsen the prognosis of prostatic cancer.

Increased neuropil antibody prevalence in COVID-19 patients with acute ischemic stroke

ABSTRACT Objectives COVID-19 infection is associated with an increased risk of acute ischemic stroke (AIS). Although the underlying mechanisms are largely unknown, autoimmunity has been implicated as a potential role player. Methods To investigate the presence and clinical impact of neuronal cell surface antibodies in COVID-19 associated AIS, patients with COVID-19 pneumonia and AIS (n = 30), COVID-19 pneumonia without AIS (n = 32) and AIS without COVID-19 infection (n = 27) were recruited. Serum anti-neuronal antibodies directed against well-characterized and novel cell surface antibodies were evaluated by cell-based assays and indirect immunohistochemistry, respectively. Results None of the recruited patients displayed well-characterized neuronal cell surface antibodies. Ten patients in the COVID-19 pneumonia with AIS group and three patients in the COVID-19 pneumonia without AIS group exhibited antibodies to neuropil of hippocampus and cerebellum. Neuropil-antibody positive patients showed trends towards milder clinical severity and reduced blood levels of inflammation factors. Conclusion Our results confirm the presence of neuropil antibodies in patients with COVID-19 infection and identify a putative antibody-driven association between AIS and COVID-19. The antigenic targets and potential pathogenic action of these antibodies need to be further explored.

Myoclonus in patients with COVID-19: Findings of autoantibodies against brain structures in cerebrospinal fluid.

COVID-19 is associated with multiple neurological manifestations. The clinical presentation, trajectory, and treatment response for three cases of myoclonus during COVID-19 infection, with no previous neurological disease, are decsribed. Analysis of cerebrospinal fluid from the cases using indirect immunohistochemistry. Antibodies against rodent brain tissue, and similarities in staining patterns were observed, indicating the presence of antineuronal immunoglobulin G autoantibodies targeting astrocytes in the hippocampus. Our results demontrate cerebrospinal fluid antineuronal antibodies indicating an an autoimmune involvment in the pathogenesis in COVID-19 associated myoclonus.

Quantification of Analgesic and Anti-Inflammatory Lipid Mediators in Long-Term Cryopreserved and Freeze-Dried Preserved Human Amniotic Membrane.

The aim of this study was to compare concentrations of endogenous N-acylethanolamine (NAE) lipid mediators-palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and anandamide (AEA)-in fresh, decontaminated, cryopreserved, and freeze-dried amniotic membrane (AM) allografts, thereby determining whether AM's analgesic and anti-inflammatory efficiency related to NAEs persists during storage. The concentrations of NAEs were measured using ultra-high-performance liquid chromatography-tandem mass spectrometry. Indirect fluorescent immunohistochemistry was used to detect the PEA PPAR-α receptor. The concentrations of PEA, OEA, and AEA were significantly higher after decontamination. A significant decrease was found in cryopreserved AM compared to decontaminated tissue for PEA but not for OEA and AEA. However, significantly higher values for all NAEs were detected in cryopreserved samples compared to fresh tissue before decontamination. The freeze-dried AM had similar values to decontaminated AM with no statistically significant difference. The nuclear staining of the PPAR-α receptor was clearly visible in all specimens. The stability of NAEs in AM after cryopreservation was demonstrated under tissue bank storage conditions. However, a significant decrease, but still higher concentration of PEA compared to fresh not decontaminated tissue, was found in cryopreserved, but not freeze-dried, AM. Results indicate that NAEs persist during storage in levels sufficient for the analgesic and anti-inflammatory effects. This means that cryopreserved AM allografts released for transplant purposes before the expected expiration (usually 3-5 years) will still show a strong analgesic effect. The same situation was confirmed for AM lyophilized after one year of storage. This work thus contributed to the clarification of the analgesic effect of NAEs in AM allografts.

BDNF and Krox20 as Indicators of Platelet-rich Plasma-Induced Nerve Regeneration in a Neuropathic Orofacial Pain Model.

Various growth factors contained in PRP can increase angiogenesis and cell proliferation, which plays an essential role in the process of neuroregeneration and peripheral nerve injury recovery. This study analyzed PRP effects in the neuro-regeneration of axonotmesis through brain-derived neurotrophic factor (BDNF) and Krox20 expressions. Freeze-dried allogeneic platelet-rich plasma (PRP) were prepared from allogeneic sources. Forty-two Rattus norvegicus were divided into three groups: negative control group, positive control group (crushing infraorbital nerve) and treatment group (crushing infraorbital nerve without PRP injection). Each group was observed for fourteen and twenty-one days after injury. Infraorbital nerve tissue is isolated for indirect immunohistochemistry examination with BDNF and Krox20 antibodies. Data analysis was performed using One-Way ANOVA and Mann-Whitney tests with significant value as p < 0.05. The PRP group showed BDNF expression significantly higher than control positive groups, both observation days (p = 0.00). A higher Korx20 expression showed by the PRP group after 21 days than in the control positive groups (p = 0.002). PRP can potentially improve neuroregeneration of axonotmesis through increased BDNF and Krox20 expression on the twenty-one days after injury.

An In-Vivo Study of Effects of Platelet-Rich Plasma on Transforming Growth Factor-β1 and Matrix Metalloprotein 9 Expression in Traumatic Ulcers with Diabetes Mellitus.

Diabetes mellitus is not only characterized by alterations in the wound healing process but also during oral ulcer healing. The platelet-rich plasma (PRP) can be used to stimulate the healing process. This study was performed to analyze the effect of PRP on traumatic ulcers with diabetes mellitus in an animal model by analyzing the expression of transforming growth factor β1 (TGF-β1) and matrix metalloprotein 9 (MMP-9). The diabetes mellitus model was developed using streptozotocin that was administrated to Rattus novergicus. The traumatic ulcer model was obtained by placing a heated tip of a ball burnisher for 5 seconds on the lower mucosa labial. Then, the traumatic ulcer was treated with PRP for 3, 5, and 7 days. The expression of TGF-β1 and MMP-9 was analyzed with indirect immunohistochemistry, and differences between each marker were analyzed with statistical analysis. All animals showed clinical oral ulceration as a yellow base during the experiment. The application of PRP showed a higher level of TGF-β1 expression than the controls for 3, 5, and 7 days (p < 0.05). In contrast, the MMP-9 expression was lower than the control for 5 and 7 days (p < 0.05). The PRP affected traumatic ulcers with diabetes mellitus by promoting healing through TGF-β1 expression and suppressing the MMP-9 expression. This material can serve to develop a promising topical therapy for traumatic ulcers, especially with an underlying disease such as diabetes mellitus.

Cerebellar antibodies in post-stroke sera of acute ischemic stroke patients

<p>Although serum anti-neuronal antibodies are found in acute ischemic stroke (AIS) patients, it is not completely clear whether they are already present before the cerebrovascular event or emerge thereafter.&nbsp;</p>. <p>Sera of 21 consecutive first-ever AIS patients were collected within the first day of AIS (baseline), as well as 1 and 6 months after AIS. Well-characterized and novel anti-neuronal antibodies were investigated by cell-based assays, immunoblotting and indirect immunohistochemistry.</p>. <p>None of the AIS sera collected at different time points showed well-characterized antibodies. In 7 patients, 1- and 6-month sera (but not baseline sera) showed IgG mostly reacting with soma and dendrites of cerebellar Purkinje cells. Antibody-positive patients did not differ in terms of clinical and etiological features.</p>. <p>Our results provide evidence for the antibody-triggering action of AIS. Although anti-cerebellar antibodies are not associated with the severity of stroke, they may potentially contribute to chronic post-stroke complications and disability.</p>.

Using Comparative Proteomics to Identify Protein Signatures in Clear Cell Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is one of the most commonly diagnosed cancers in the world. Approximately 25-30% of patients identified with initial kidney cancer will have metastasized tumors, thus 5-year survival rates for these patients are poor. Therefore, biomarker research is required to identify and predict molecular signatures in RCC. To address this, we used a mass spectrometry (MS)-based proteomics approach to identify proteins related to clear cell RCC (ccRCC) tissues from patients with T1G2, T1G3, T3G2, T3G3, and metastatic RCC (mRCC) stages. We identified and quantified 2,608 and 2,463 proteins, respectively, in ccRCC tissue and identified 1,449 differentially expressed proteins (DEPs). Bioinformatics analysis revealed that serpin family A member 3 (SERPINA3) qualified as biomarker for ccRCC progression. Using indirect enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunohistochemistry assays it was found that SERPINA3 expression levels in ccRCC tissues were much higher in stages before metastasis. Comparative proteomics analysis of ccRCC tissues provided new evidence of SERPINA3 association with ccRCC progression.

The diagnosis of anti-LGI1 encephalitis varies with the type of immunodetection assay and sample examined.

Detection of Leucine-rich glioma inactivated 1 (LGI1) antibodies in patients with suspected autoimmune encephalitis is important for diagnostic confirmation and prompt implementation of immunomodulatory treatment. However, the clinical laboratory diagnosis can be challenging. Previous reports have suggested that the type of test and patient's sample (serum or CSF) have different clinical performances, however, there are no studies comparing different diagnostic tests on paired serum/CSF samples of patients with anti-LGI1 encephalitis. Here, we assessed the clinical performance of a commercial and an in house indirect immunofluorescent cell based assays (IIF-CBA) using paired serum/CSF of 70 patients with suspected anti-LGI1 encephalitis and positive rat brain indirect immunohistochemistry (IIHC). We found that all (100%) patients had CSF antibodies when the in house IIF-CBA was used, but only 88 (83%) were positive if the commercial test was used. In contrast, sera positivity rate was higher with the commercial test (94%) than with the in house assay (86%). If both serum and CSF were examined with the commercial IIFA-CBA, 69/70 (98.5%) patients were positive in at least one of the samples. These findings are clinically important for centers in which rat brain IIHC and in house IIFA-CBA are not available. Moreover, the observation that all patients with anti-LGI1 encephalitis have antibodies in CSF is in line with the concept that these antibodies are pathogenic.

SSEA-4 positive dental pulp stem cells from deciduous teeth and their induction to neural precursor cells

BackgroundStage-specific embryonic antigen-4 (SSEA-4) is a marker for the identification of multipotent embryonic cells. It is also positive in neuroepithelial cells, precursor neural cells (NPC), and human dental pulp cells. The aim of this study was to evaluate the potential morphodifferentiation and histodifferentiation to NPC of SSEA-4 positive stem cells from human exfoliated deciduous teeth (SHED).MethodsA SHED population in culture, positive to SSEA-4, was obtained by magnetic cell separation. The cells were characterized by immunohistochemistry and flow cytometry. Subsequently, a neurosphere assay was performed in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF); afterward, cells were neurodifferenciated with a neurobasal medium. Finally, indirect immunohistochemistry was performed to identify neuronal markers.ResultsThe morphological and histological changes in the SSEA-4 positive SHEDs were observed after induction with epidermal and fibroblast growth factors in neurobasal culture medium. At the end of induction, the markers Nestin, TuJ-1, and GFAP were identified.ConclusionsThe findings show that SSEA-4 positive SHEDs have a behavior similar to neuronal precursor cells. Our findings indicate that the dental pulp of deciduous teeth is a promising source for regeneration therapies associated with neurodegenerative diseases or peripheral nerve alterations.

Detection of Glycosaminoglycans in Pancreatic Islets and Lymphoid Tissues.

In this chapter, we describe the detection of the glycosaminoglycans hyaluronan and heparan sulfate in pancreatic islets and lymphoid tissues. The identification of hyaluronan in tissues is achieved by utilizing a highly specific hyaluronan binding protein (HABP) probe that interacts with hyaluronan in tissue sections. The HABP probe is prepared by enzymatic digestion of the chondroitin sulfate proteoglycan aggrecan which is present in bovine nasal cartilage and is then biotinylated in the presence of bound hyaluronan and the link protein. Hyaluronan is then removed by gel filtration chromatography. The biotinylated HABP-link protein complex is applied to tissue sections, and binding of the complex to tissue hyaluronan is visualized by enzymatic precipitation of chromogenic substrates.To determine hyaluronan content in tissues, tissues are first proteolytically digested to release hyaluronan from the macromolecular complexes that this molecule forms with other extracellular matrix constituents. Digested tissue is then incubated with HABP . The hyaluronan-HABP complexes are extracted, and the hyaluronan concentration in the tissue is determined using an ELISA-like assay.Historically, heparan sulfate was identified in tissue sections using the cationic dye Alcian blue and histochemistry based on the critical electrolyte concentration principle of differential staining of glycosaminoglycans using salt solutions. For both human and mouse pancreas sections, the current optimal method for detecting heparan sulfate is by indirect immunohistochemistry using a specific anti-heparan sulfate monoclonal antibody. A peroxidase-conjugated secondary antibody is then applied, and its binding to the anti-heparan sulfate antibody is visualized by oxidation and precipitation of a chromogenic substrate.

Sequential Indirect Dual Immunohistochemistry with Primary Rabbit Antibodies on Cochlear Sections Using an Intermediate Heat-Denaturation Step.

Advanced immunohistochemical (IHC) protocols aim to visualize different molecules in situ simultaneously. These techniques are of utmost importance as a first step in studying possible interactions of proteins at the subcellular level. Colocalized stains in tissue sections indicate proximity of two proteins of interest. Frequently, double staining protocols are restricted by the lack of primary antibodies generated in different animal species for indirect IHC visualization. Here, we present a detailed protocol for mouse inner ear tissue using two different primary rabbit antibodies directed against transmembrane ion channel proteins of cochlear neurons. The two antibodies are combined for fluorescence (confocal) as well as dual multiplex colorimetric visualization in two sequential single IHC stainings. A heat-denaturation step is performed in between. Primary antibody specificity is tested by preadsorption with the immunogenic peptide, and positive and negative tissue controls are performed to confirm the reliability of the antibody detection. We describe the whole procedure in detail beginning with tissue extraction of the mouse inner ear and continuing with chemical fixation, cryoembedding, and preparation for manual and fully automated immunostaining, including steps for heat-induced antigen retrieval. The potential to use antibodies from the same host species for single and double IHC staining opens up multiple possibilities for detecting different targets in the same tissue section using resources and materials that are widely available. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tissue preparation, cryoembedding, and sectioning Basic Protocol 2: Double colorimetric immunostaining with an automatic immunostainer Basic Protocol 3: Double manual fluorometric immunostaining with fluorescence.