Post-fixation Step Research Articles

Cutaneous T-Cell Lymphoma (CTCL) Cell Line-Derived Extracellular Vesicles Contain HERV-W-Encoded Fusogenic Syncytin-1

Cutaneous T-Cell Lymphoma (CTCL) Cell Line-Derived Extracellular Vesicles Contain HERV-W-Encoded Fusogenic Syncytin-1

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

Protocol for HER2 FISH determination on PAXgene-fixed and paraffin-embedded tissue in breast cancer.

SummaryMolecular diagnostics in personalized medicine increasingly relies on the combination of a variety of analytical technologies to characterize individual diseases and to select patients for targeted therapies. The gold standard for tissue‐based diagnostics is fixation in formalin and embedding in paraffin, which results in excellent preservation of morphology but negatively impacts on a variety of molecular assays. The formalin‐free, non‐cross‐linking PAXgene tissue system preserves morphology in a similar way to formalin, but also preserves biomolecules essentially in a similar way to cryopreservation, which markedly widens the spectrum, sensitivity and accuracy of molecular analytics. In this study, we have developed and tested a protocol for PAXgene‐fixed and paraffin‐embedded tissues for fluorescent in situ hybridization (FISH). The implementation of a 24‐h formalin postfixation step of slides from PAXgene‐fixed and paraffin‐embedded tissues allowed us to use the assays approved for formalin‐fixed and paraffin‐embedded tissues. The equivalence of the methodologies was demonstrated by FISH analysis of HER2 amplification in breast cancer cases. The 24‐h postfixation step of the slides used for FISH can be well integrated in the routine diagnostic workflow and allows the remaining PAXgene‐fixed and paraffin‐embedded tissue to be used for further molecular testing.

A simple histological technique to improve immunostaining when using DNA denaturation for BrdU labelling

The typical immunohistochemistry technique used to reveal 5-bromo-2′-deoxyuridine (BrdU) incorporation requires denaturation of the DNA by heat and acid to permeabilize the cell nucleus. This treatment can damage tissue and reduce the antigenicity of several proteins, which then leads to weak immunostaining and/or false negatives. We show that an overnight post-fixation step following immunohistochemistry for antigens of interest protects immunostaining during the acid/heat denaturation treatment for subsequent BrdU staining. We used this technique to study the differentiation of recently divided oligodendrocyte progenitor cells in NG2CreER:EYFP reporter mice. We used a GFP anti-EYFP antibody to maximize visualization of the EYFP-containing oligodendrocyte progenitor cells, Olig1, and GST-pi to confirm the cell phenotype. Immunostaining for GFP, Olig1, and GST-pi is reduced by DNA denaturation. We found that incorporating a post-fixation step after double immunostaining for GFP/Olig1 and GFP/GST-pi prior to DNA denaturation prevented the fading and false negatives associated with this treatment. This simple addition to BrdU immunohistochemistry protocols extends the range of proteins that can be detected in combination with BrdU, along with the number of antibodies that can be used successfully in the study of cell proliferation.

Immunofluorescence protects RNA signals in simultaneous RNA–DNA FISH

Cell research often requires combinational detection of RNA and DNA by fluorescence in situ hybridization (RNA–DNA FISH). However, it is difficult to preserve the fragile RNA signals through the harsh conditions used to denature the DNA template in DNA FISH. The current protocols of RNA–DNA FISH still cannot work robustly in all experiments. RNA–DNA FISH remains as a technically challenging and tedious experiment. By incorporating protein components into the signal detection steps of RNA FISH, which is then followed by a post-fixation step, we established an improved protocol of RNA–DNA FISH. The established method worked satisfyingly and robustly in our studies on Xist (inactivated X chromosome specific transcript) RNA and Terra (telomeric repeat-containing RNA). Our results provided the direct evidence to show that, not all the telomeres are associated with Terra, and a significant fraction of Terra foci do not overlap with telomere DNA in interphase cell nuclei. The improved method of simultaneous RNA–DNA FISH is reliable and time-efficient. It can be used in a variety of biological studies.

Effects of Ethanol Post-Fixation on the Histological, Histochemical, and Immunohistochemical Analysis of Murine Bone Specimens

Proper histological and histomorphometric assessment of bones requires acute visualization of cellular detail. Because fat is soluble in ethanol, adding an ethanol post-fixation step can help remove excess fat in tissue specimens, thus enhancing cellular visualization. However, the effects of adding this additional ethanol post-fixation step on bone-specific histochemical and immunohistochemical protocols have not yet been elucidated. Here, we describe the differences between using or not using an additional ethanol post-fixation step on six types of histochemical and immunohistochemical protocols. We assessed the effects in undecalcified tibiae sections routinely stained with hematoxylin and eosin, toluidine blue, and Von Kossa and found that addition of ethanol post-fixation enhanced specimen visualization for each of these stains. We examined the of ethanol post-fixation on enzymatic staining and found that alkaline phosphatase activity was equally preserved, whereas acid phosphatase activity was visualized more readily in specimens that underwent this treatment. Finally, the ethanol post-fixation improved antigen preservation in osteocalcin immunohistochemical labeling. (The J Histotechnol 27: 15, 2004)Submitted: July 8, 2003; Accepted: October 31, 2003

Image Analysis for Quantitative Evaluation of Antigen Retrieval Efficacy Demonstrates Increased Detection of P-glycoprotein in Overfixed Cells but Decreased Detection in Optimally Fixed Cells

We developed a protocol to objectively quantify, via image analysis, the results of antigen retrieval (AR) maneuvers on immunohistochemical results for detection of P-glycoprotein (P-gp) in paraffin embedded test samples. Two carcinoma cell lines, KB 3-1 and KB V-1 (previously described non-expresser and high expresser of P-glycoprotein, respectively), were harvested, fixed for 4, 8, 12, 24 and 48 hr in formalin, placed in collodion bags, embedded into a single paraffin block, subjected to various antigen retrieval techniques, and stained with the anti-P-gp antibody C494. The amount of immunostaining was quantitated with the CAS 2.0 cell measurement program and optical density ratios were compared between the positive and negative cell lines. In overfixed cells (48 hr, 10% formalin), AR with microwaves and/or trypsin or other enzymes proved effective in optimizing P-gp detection as evidenced by an increased optical density ratio. In contrast, quantitation of staining of optimally fixed cells (4 hr in 10% formalin) demonstrated that AR decreased the optical density ratio. A postfixation step after the primary antibody also served to decrease the optical density ratio, via an increase in noise in the non-expressing cell. The use of optical density analysis by image computerization allowed objective, exquisitely sensitive, quantitative assessment of AR methods. (The J Histotechnol 22:9, 1999)

Image Analysis for Quantitative Evaluation of Antigen Retrieval Efficacy Demonstrates Increased Detection of P-glycoprotein in Overfixed Cells but Decreased Detection in Optimally Fixed Cells

We developed a protocol to objectively quantify, via image analysis, the results of antigen retrieval (AR) maneuvers on immunohistochemical results for detection of P-glycoprotein (P-gp) in paraffin embedded test samples. Two carcinoma cell lines, KB 3-1 and KB V-1 (previously described non-expresser and high expresser of P-glycoprotein, respectively), were harvested, fixed for 4, 8, 12, 24 and 48 hr in formalin, placed in collodion bags, embedded into a single paraffin block, subjected to various antigen retrieval techniques, and stained with the anti-P-gp antibody C494. The amount of immunostaining was quantitated with the CAS 2.0 cell measurement program and optical density ratios were compared between the positive and negative cell lines. In overfixed cells (48 hr, 10% formalin), AR with microwaves and/or trypsin or other enzymes proved effective in optimizing P-gp detection as evidenced by an increased optical density ratio. In contrast, quantitation of staining of optimally fixed cells (4 hr in 10% formalin) demonstrated that AR decreased the optical density ratio. A postfixation step after the primary antibody also served to decrease the optical density ratio, via an increase in noise in the non-expressing cell. The use of optical density analysis by image computerization allowed objective, exquisitely sensitive, quantitative assessment of AR methods. (The J Histotechnol 22:9, 1999)

Characterization of the proliferation state in canine mammary tumors by the standardized AgNOR method with postfixation and immunohistologic detection of Ki-67 and PCNA.

Paraffin-embedded tissue sections of 96 surgically removed mammary tumors from female dogs were analyzed for their proliferation state using three different methods. The AgNOR method, originally developed by Ploton and coworkers in 1986, modified and standardized by the AgNOR committee, is an easy, inexpensive silver-staining procedure used to determine cell proliferation and prognosis of various tumors. Due to the standardized staining protocol of the AgNOR method and a postfixation step, results obtained were of excellent quality for image-analysis processing. The growth fraction was evaluated by counting of immunohistologically positive-stained cells for Ki-67 or proliferating-cell nuclear antigen (PCNA). The values determined were, in general, lower with Ki-67 (MIB1) than with PCNA (PC10). Nevertheless, the labeling indices of these antigens correlated significantly (P < 0.001). Though the differences of the means between the tumor groups according to the classification system of the World Health Organization were significant for all three investigated methods (P < 0.001), there was a considerable overlap between the tumor groups concerning all investigated parameters. An exploratory data analysis (multivariate analysis) as performed to evaluate the prognostic relevance of the three methods including further anamnestic, clinical, gross, and histopathologic variables. Besides the histopathologic diagnosis (survival P < 0.001; survival time P < 0.05; reappearance of tumor growth P < 0.05), only the PCNA-labeling index (time until reappearance of tumor growth P < 0.001) was of prognostic significance.

Preservation of cartilage matrix proteoglycans using cationic dyes chemically related to ruthenium hexaammine trichloride.

We tested various cationic dyes chemically related to ruthenium hexaammine trichloride (RHT) [i.e., the RHT-cyclohexanedione complex (RHT-CC), pentaamine ruthenium N-dimethylphenylenediimine trichloride (PRT), tris-(bipyridyl)ruthenium (II) chloride (TRC), tris (bipyridyl) iron (II) chloride (TIC), and cobalt hexaammine trichloride (CHT)] for their effectiveness in precipitating cartilage matrix proteoglycans in situ. Dyes were introduced into media at the onset of processing and were present throughout both aldehyde fixation and osmium tetroxide post-fixation. Contrary to expectation, most of the dye-proteoglycan complexes generated and stable under aldehyde fixation conditions were found to be unstable during post-fixation despite the continuing presence of the dye. A similar phenomenon was also found for the cationic dyes commonly used for precipitation of proteoglycans in cartilage tissue sections (such as Acridine Orange, Alcian Blue, Azure A, Methylene Blue, and Ruthenium Red). Only two dyes, i.e., RHT and the newly tested RHT-CC, formed proteoglycan precipitates sufficiently stable to resist disruption and extraction during osmium tetroxide post-fixation. The latter may be particularly useful in semiquantitative analyses of proteoglycan content in unstained tissue sections owing to its intense brown-black color. For applications in which the osmium tetroxide post-fixation step may be omitted, TRC and PRT may also be valuable for semiquantitative histochemistry by virtue of their stable fluorescence and intense violet color signals, respectively.

Staining terminal cisternae proteins in rat skeletal muscle triads: Selectivity of a new silver technique

A selective silver staining of proteins within the lateral components of triads and diads in fixed striated muscle cells and in isolated sarcoplasmic reticulum vesicles using a mercuric acetate postflxation has been demonstrated by us.In the present work we modify the technique in order to study the mechanism of the reaction . First, the mercuric postfixation step was omitted and replaced by a treatment with diluted ammonia, a procedure thought to wash out the excess of the reducing aldehyde fixative. Second, a treatment with mercuric acetate was employed on the ammonia treated muscles. The rat gastrocnemius muscle was fixed by immersion (12-24 h) in 3% glutaraldehyde, washed briefly with water and placed (12-24 h) into an ammonia:distilled water (1:4 v/v) solution. After washing with water, 1% acetic acid and water, muscles were treated overnight with 50% (v/v) ammoniacal silver nitrate at 37°C in darkness, washed with three changes of 5% (w/v) sodium sulphite and water, embedded in Poly B, sectioned and examined unstained with a Siemens Elmiskop I electron microscope.

Silver staining of interphase nucleolar organizer regions in cytologic smears previously stained by the papanicolaou and may‐Grünwald‐Giemsa Techniques

Malignant cells are known to display a distinctive argyrophilic nucleolar organizer region (Ag-NOR) protein distribution compared with distribution in their benign counterparts. In this study, Ag-NOR staining was applied to smears from reactive and malignant serous effusions. The smears were either unstained or had been previously stained with the Papanicolaou or May-Grünwald-Giemsa (MGG) technique. The purpose of this study was to assess the feasibility and usefulness of Ag-NOR staining in diagnostic cytopathology and to set up a procedure that could be used on prestained smears in retrospective studies. The one-step silver staining method was applied to prestained smears after a weak destaining step in trichloracetic acid and to unstained smears after a postfixation step in Carnoy's fluid. Positive results were observed on both destained and unstained preparations, MGG-stained smears showing a better visualization of the silver deposits than Papanicolaou-stained smears. Major differences in size, shape, and distribution patterns of the Ag-NOR-positive granules were observed between neoplastic and reactive cells. Furthermore, the mean number of silver-stained dots per cell was significantly higher in malignant cells (33.32 +/- 2.23) than in reactive mesothelial cells (9.71 +/- 0.66). These data indicate that this Ag-NOR technique can be profitably applied to prestained cytologic smears to assist in the diagnosis of malignancy and that the technique has the advantage that cellular morphology and silver staining can be evaluated on the same slide.

The effect of digitonin-containing fixatives on the retention of free cholesterol and cholesterol esters.

The influence of several fixation and dehydration procedures on the retention of free cholesterol and cholesterol esters was studied in filter paper preparations. The retention of free cholesterol by the filter paper proved to be decreased by the addition of digitonin to the aldehyde fixative (aqueous phase) and was only slightly enhanced by partial dehydration (alcoholic phase, up to 70% ethanol). Furthermore, digitonin or the presumably formed cholesterol-digitonide complex bound hardly any osmium oxides in glass-fibre paper. Up to 26% of the cholesterol esters was mobilized during the aqueous phase when digitonin was added to the aldehyde fixative. When the glass-fibre papers containing the digitonin cholesterol-ester-osmate complexes were stored in distilled water after fixation, the fluid became turbid. Particulate material isolated from this turbid solution showed ultrastructurally a close resemblance to the 'whorls' observed by several authors in tissue fixed by a digitonin-containing aldehyde fixative. Digitonin also changed the ultrastructural appearance of liposomes, containing lecithin: cholesterol: phosphatidic acid; in a molar ratio 7:2:1. Our observations lead to the conclusion that the use of digitonin-containing fixatives should be abandoned, because they give results which cannot be interpreted. By the use of K4 [Fe(CN)6] containing OSO4 in the post-fixation step were able to demonstrate an increase in the visualization of membranous structures (liposomes).

Optimization of the binding of dissociated exfoliated cervico-vaginal cells to glass microscope slides.

In order to monitor the development of a cell dissociation technique, it was essential to utilize the Centrifugal Cytology rotor to produce glutaraldehyde-fixed even cellular dispersions. The Cytology rotor has been improved to insure rapid alignment with the centrifugal field during both acceleration and deceleration, and the fixative is now delivered to the surface of the slide. The dissociation of the cells results in a loss of their adhesion to glass slides. Three bonding agents were tested: (a) Poly-L-Lysine; (b) Mayer's albumin fixative; (c) positively charging the slides with a silicone coating. The results with 65% albumin-coated slides were clearly superior to the other two. The addition of a postfixation step of 95% ethanol/4% polyethylene glycol did not significantly affect the recovery of the cells, but did eliminate some unevenness in the Centrifugal Cytology preparations, flattened the cells and expedited the procedure.