Indirect Immunocytofluorescence Research Articles

Distribution and expression of α-actin-4 in puromycin aminonueleoside injured mouse podocyte cell line

Objective To observe the expression and distribution of α-actin-4 mRNA through puromycin aminonueleoside(PAN) injury podocyte, and discuss the relation between α-actin-4 and podocyte damage. Methods Podocytes were cultured in vitro, and 2 groups were set up: control group and PAN stimulation group.The control group was cultured with concentration of 100 mL/L FBS RPMI 1640 nutrient solution culture, while the PAN group was cultiva-ted to PAN(50 mg/L) treatment, and cell morphology, extraction of total RNA of α-actin-4 were observed in 8 h, 24 h and 48 h. The podocyte morphology was observed and pictures were taken through phase-contrast microscope, then the diffe-rences of morphology and areas between the 2 groups were analyzed.The distribution and mRNA expression of α-actin-4 were detected by indirect immunocyto-fluorescence and real-time quantitative PCR, respectively. Results The well-deve-loped podocyte arborization was formed after the in vitro induction, and the PAN treatment led to the podocyte foot process retraction and effacement together with the mouse podocyte cell line shrinkage and the loss of cell contact.The above time point α-actin-4 mRNA expressions between the 2 groups were compared, and there was no significant difference in 8 h(P>0.05), but significant difference was found in 24 h, 48 h, α-actin-4 higher mRNA expression, with statistical significance(all P<0.01). α-actin-4 in the control group had thin filaments evenly distributed in the cytoplasm, but a radioa-ctive distribution in foot process.In the experimental group, α-actin-4 pressure silk fiber was shorter, with disordered arrangement, and PAN stimulus after 24 h, α-actin-4 distribution in cytoplasm was decreased significantly, while cytoplasmic distribution was missing after 48 h. Conclusions The abnormal of distribution and mRNA expression of α-actin-4 is time-related to the PAN injury podocyte, and α-actin-4 is an important part of podocyte damage mechanism. Key words: Podocyte; α-actin-4; Puromycin aminonueleoside

Persistent Infection of a Carcinoma Thyroid Cell Line with Coxsackievirus B

Viral infections are described as environmental factors that are implicated in various thyroid diseases. The role of enteroviruses (EV) in the pathogenesis of thyroid diseases has been suspected. Recently, we found that EV RNA could be detected in postoperative thyroid specimens. We decided to investigate the infection of a human thyroid cell line with coxsackievirus B4 (CVB4). The wild-type human thyroid carcinoma cells K1 were inoculated with CVB4E2 at 2.1 x 10(7) TCID50/mL. The titer of the virus progeny was determined every 3 days on HEp-2 cells. CVB replication at the molecular level was monitored by searching for intracellular viral genomes using reverse transcription (RT)-polymerase chain reaction (PCR). EV VP1 capsid protein was detected by indirect immunocytofluorescence staining. Cell viability was determined by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) absorbance, and the nuclear morphology was assessed by Hoechst Dye staining. Infectivity assays with CVB4E2 revealed an increase in viral titers. Virus production decreased thereafter, but was not stopped by serial subculture for 24 days after infection. Detection of intracellular positive and negative viral RNA strands by RT-PCR was positive between days 1 and 14 postinfection (p.i.), and by semi-nested RT-PCR up to 24 days. K1 cell cultures infected with CVB4 were stained positively for EV VP1: the number of VP1-positive cells decreased rapidly within 6 days and remained low up to the end of culture. Compared with mock-infected cultures, viability in CVB4-infected cultures was around 100% up to 24 days. Cells with strongly fluorescent nuclei and/or fragmented nuclei were observed. We demonstrate for the first time that CVB4 could replicate in thyroid cells and could persist, with predominance of viral negative RNA strands for up to 24 days p.i. without obvious cytopathic effect. Our results suggest that CVB4 may lead to thyroid cell apoptosis. Further studies are needed to determine whether CVB could play a role in thyroid pathologies.

Localization of Functional Prostaglandin E2 Receptors EP3 and EP4 in the Nuclear Envelope

The effects of prostaglandin E2 are thought to be mediated via G protein-coupled plasma membrane receptors, termed EP. However recent data implied that prostanoids may also act intracellularly. We investigated if the ubiquitous EP3 and the EP4 receptors are localized in nuclear membranes. Radioligand binding studies on isolated nuclear membrane fractions of neonatal porcine brain and adult rat liver revealed the presence of EP3 and EP4. A perinuclear localization of EP3alpha and EP4 receptors was visualized by indirect immunocytofluorescence and confocal microscopy in porcine cerebral microvascular endothelial cells and in transfected HEK 293 cells that stably overexpress these receptors. Immunoelectron microscopy clearly revealed EP3alpha and EP4 receptors localization in the nuclear envelope of endothelial cells; this is the first demonstration of the nuclear localization of these receptors. Data also reveal that nuclear EP receptors are functional as they affect transcription of genes such as inducible nitric-oxide synthase and intranuclear calcium transients; this appears to involve pertussis toxin-sensitive G proteins. These results define a possible molecular mechanism of action of nuclear EP3 receptors.

CDNA Cloning, Expression, Mutagenesis, Intracellular Localization, and Gene Chromosomal Assignment of Mouse 5-Lipoxygenase

5-Lipoxygenase of mouse macrophages and bone marrow-derived mast cells (BMMC) was investigated. Indirect immunocytofluorescence combined with confocal microscopy provided evidence for distinct intracellular expression patterns and trafficking of 5-lipoxygenase upon cellular activation. In resting BMMC, 5-lipoxygenase was found within the nucleus co-localizing with the nuclear stain Yo-Pro-1. When BMMC were IgE/antigen-activated the 5-lipoxygenase immunofluorescence pattern was changed from nuclear to perinuclear. The absence of divalent cations in the incubation medium, or calcium ionophore A23187 challenge, altered the predominantly nuclear expression pattern to new sites both cytosolic and intranuclear. The cDNA for murine macrophage 5-lipoxygenase was cloned by the polymerase chain reaction and would predict a 674 amino acid protein. Using control cells obtained from 5-lipoxygenase-deficient mice it was determined that a single isoform accounts for both soluble and membrane-bound and nuclear and cytosolic-localized enzyme in macrophages and BMMC. A mutation at amino acid 672 (Val-->Met) introduced serendipitously during the cloning process was found to completely abolish 5-lipoxygenase enzyme activity when the enzyme was expressed in human embryonic kidney 293 cells. This subtle change is proposed to affect the ability of the COOH-terminal isoleucine to coordinate the essential non-heme iron atom. In macrophages and BMMC obtained from 5-lipoxygenase-deficient mice, compensatory changes in expression of genes involved in the biosynthesis of leukotriene B4 were investigated. 5-Lipoxygenase-activating protein expression was reduced by 50%, while leukotriene A4 hydrolase expression was unaltered. The 5-lipoxygenase gene was mapped to the central region of mouse chromosome 6 in a region that shares homology with human chromosome 10 by interspecific backcross analysis. These studies provide a global picture of the murine 5-lipoxygenase system and raise questions about the role of 5-lipoxygenase and leukotrienes within the nucleus.

Differential localization of protein kinase C isozymes in U937 cells: evidence for distinct isozyme functions during monocyte differentiation

U937 human promonocytic leukemia cells express PKC isozymes beta 1, beta 2, epsilon and zeta. Indirect immunocytofluorescence using affinity-purified PKC-specific antibodies indicates that each of the endogenous PKC isozymes in U937 cells display a unique compartmentalization within the intact cell. PKC-beta 1 is distributed between two identifiable pools: a cytoplasmic pool which redistributes to the plasma membrane upon activation with acute phorbol ester-treatment, and a membrane-bound pool associated with intracellular vesicles containing beta 2-integrin adhesion molecules, cd11b and cd11c. The vesicle-associated PKC-beta 1 translocates with the secretory granules to the plasma membrane upon agonist-stimulated activation. PKC-beta 2 is associated with the microtubule cytoskeleton in resting cells. PKC overlay assays indicate that PKC-beta 2 binds to proteins associated with microtubules, and not directly to tubulin. PKC-epsilon is associated with filamentous structures in resting cells and redistributes to the perinuclear region upon activation with phorbol esters. In differentiated U937 cells, PKC-beta 1 remains associated with vesicles translocating from the trans-Golgi region to the plasma membrane and PKC-epsilon is primarily associated with perinuclear and plasma membranes. PKC-zeta, which does not respond to phorbol ester treatment, is primarily cytosolic in undifferentiated cells and accumulates in the nucleus of differentiated cells blocked in the G2 phase of the cell cycle. The data clearly demonstrate that individual PKCs localize to different subcellular compartments and promote the hypothesis that PKC subcellular localization is indicative of unique functions for individual PKC isozymes.

Changes in the localization of catalase during differentiation of neutrophilic granulocytes

The nature of the compartmentalization of catalase in human myeloid cells is an unresolved issue. Using a rabbit polyclonal antibody specific for catalase, indirect immunocytofluorescence of immature leukemic promyelocytes (HL-60 cells) showed a pattern of small, sharp, punctate staining in the cytoplasm of all cells, while mature neutrophils showed a larger diffuse, flocculent pattern of cytoplasmic staining. Differential centrifugation of nitrogen cavitates of HL-60 cells indicated that the putative catalase-containing compartment was relatively fragile compared with the compartment(s) that contained myeloperoxidase (MPO), beta-hexosaminidase, beta-glucuronidase, and lysosomal alpha-mannosidase activities. Parallel studies using dimethylsulfoxide (DMSO)-induced HL-60 cells and mature neutrophils showed that, in the course of differentiation, there was an apparent shift in the localization of catalase from the granule fraction to the cytosolic fraction. Percoll-sucrose density gradient centrifugation of HL-60 cell cavitates showed a catalase-containing compartment with a mean peak density (1.05 g/mL) significantly lower than that of the major myeloperoxidase-containing compartment (1.08 g/mL); in mature neutrophils, catalase activity comigrated with lactate dehydrogenase (LDH) activity. Catalase in isolated fractions was protected from proteolysis in the absence, but not in the presence, of 0.1% Triton X-100. Digitonin titration experiments confirmed the compartmentalized nature of catalase in immature HL-60 cells and were consistent with a cytosolic localization in mature neutrophils. Ultrastructural localization of catalase by Protein A-gold immunocytochemistry demonstrated four to six catalase-containing compartments in all HL-60 cell profiles. In mature neutrophils, catalase was localized primarily in the cytoplasmic matrix, although in fewer than 2% of the cell profiles, one to two catalase-containing compartments were observed. The changes in catalase localization that occur during myeloid differentiation appear to be similar to the changes that occur during erythroid and megakaryocytic differentiation, and may have potential clinical significance in the classification of acute leukemia and in the development of drug resistance.

Changes in the Localization of Catalase During Differentiation of Neutrophilic Granulocytes

AbstractThe nature of the compartmentalization of catalase in human myeloid cells is an unresolved issue. Using a rabbit polyclonal antibody specific for catalase, indirect immunocy-tofluorescence of immature leukemic promyelocytes (HL-60 cells) showed a pattern of small, sharp, punctate staining in the cytoplasm of all cells, while mature neutrophils showed a larger diffuse, flocculent pattern of cytoplasmic staining. Differential centrifugation of nitrogen cavitates of HL-60 cells indicated that the putative catalase-containing compartment was relatively fragile compared with the compart-ment(s) that contained myeloperoxidase (MPO), β-hexos-aminidase, β-glucuronidase, and lysosomal α-mannosidase activities. Parallel studies using dimethylsutfoxide (DMSO)-induced HL-60 cells and mature neutrophils showed that, in the course of differentiation, there was an apparent shift in the localization of catalase from the granule fraction to the cytosolic fraction. Percoll-sucrose density gradient centrifugation of HL-60 cell cavitates showed a catalase-containing compartment with a mean peak density (1.05 g/mL) significantly lower than that of the major myeloperoxidase-containing compartment (1.08 g/mL); in mature neutrophils, catalase activity comigrated with lactate dehydrogenase (LDH) activity. Catalase in isolated fractions was protected from proteolysis in the absence, but not in the presence, of 0.1 % Triton X-100. Oigitonin titration experiments confirmed the compartmentalized nature of catalase in immature HL-60 cells and were consistent with a cytosolic localization in mature neutrophils. Ultrastructural localization of catalase by Protein A-gold immunocytochemistry demonstrated four to six catalase-containing compartments in all HL-60 cell profiles. In mature neutrophils, catalase was localized primarily in the cytoplasmic matrix, although in fewer than 2% of the cell profiles, one to two catalase-containing compartments were observed. The changes in catalase localization that occur during myeloid differentiation appear to be similar to the changes that occur during erythroid and megakaryocytic differentiation, and may have potential clinical significance in the classification of acute leukemia and in the development of drug resistance.

Formula omitted] expression in natural killer (NK) cells enriched from peripheral or umbilical cord blood

The sensitivity to antineoplastic agents of subpopulations of haematopoietic cells during cancer chemotherapy is an open question. The performance of natural killer (NK) cells, possibly assisting the elimination of tumour cells under drug treatment might be of particular interest. We examined the expression of the transmembrane multidrug transporter mdr1 P - glycoprotein in NK-cells (CD56 +) enriched from the peripheral blood or the umbilical cord blood from healthy donors by indirect immunocytofluorescence using the monoclonal P-glycoprotein antibody C219 and a polymerase chain reaction (PCR) approach with amplimers specific for the human mdr1 cDNA. As the antibody C219 apparently cross-reacts with the human mdr3 gene product whose functions are as yet unclear we also checked expression of this gene by PCR using mdr3 specific amplimers. Distinct, but rather inhomogeneous mdr1 P - glycoprotein expression was found in NK-cells enriched from the peripheral blood. NK-cells enriched from the umbilical cord blood showed quite strong mdr1 expression levels throughout, exceeding the values found in the moderately multidrug-resistant cell line CCRF VCR 100 which is permanently cultivated in the presence of 100 ng/ml vincristine. Mdr1 P - glycoprotein expression was mirrored by lowered sensitivities of the cultivated NK-cells towards actinomycin D or adriamycin. The drug sensitivity could be modulated by treatment of the cells with the immunosuppressive drug cyclosporin A. Expression of the mdr3 gene was low or absent in all NK-cell samples examined so far.

Subcellular Localization of Prostaglandin Endoperoxide Synthase-2 in Murine 3T3 Cells

Polyclonal antisera specific for prostaglandin endoperoxide (PGH) synthases-1 and -2 were used to determine the subcellular locations of each PGH synthase isozyme in detergent-permeabilized mouse 3T3 fibroblasts by indirect immunocytofluorescence. Antiserum to PGH synthase- 1 demonstrated a mottled pattern of cytoplasmic and perinuclear staining of both serum-starved and serum-stimulated 3T3 cells. This pattern of staining is consistent with the results of earlier studies which demonstrated that PGH synthase-1 is associated with the endoplasmic reticulum and nuclear envelope of these cells. As expected, antibodies directed against a peptide unique to PGH synthase-2 failed to stain serum-starved cells, which lack appreciable levels of this second form of the enzyme. However, serum-stimulated 3T3 cells, which do express PGH synthase-2, showed the same pattern of staining with PGH synthase-2 antibodies as was observed with anti-PGH synthase-1 serum-mottled cytoplasmic staining and perinuclear staining. We conclude that the subcellular location of PGH synthase-2 is the same as PGH synthase-1 in murine 3T3 cells. Thus, the notable differences in the primary amino acid sequence-the signal peptide and the additional 18 amino acid C-terminal segment in PGH synthase-2-do not cause a change in localization. Colocalization of PGH synthases-1 and -2 implies that the source of arachidonate substrate, the site of PGH 2 and prostanoid formation, and the mechanism of product transport from the inside to the outside of the cell are the same for these isozymes.

Mdr1/P-glycoprotein, topoisomerase, and glutathione-S-transferase pi gene expression in primary and relapsed state adult and childhood leukaemias.

In a variety of adult and childhood leukaemia cell samples collected at different states of the disease, we analysed in a series of sequentially performed slot-blot or Northern-blot hybridisation experiments the expression of genes possibly involved in multiple drug resistance (MDR) (mdr1/P-glycoprotein, DNA topoisomerase II, glutathione-S-transferase pi), and the expression of the DNA topoisomerase I and histone 3.1 genes. Occasionally, P-glycoprotein gene expression was additionally examined by indirect immunocytofluorescence using the monoclonal antibody C219. No significant difference in mdr1/P-glycoprotein mRNA levels between primary and relapsed state acute lymphocytic leukaemias (ALL) was seen on average. Second or third relapses, however, showed a distinct tendency to an elevated expression of this multidrug transporter gene (up to 10-fold) in part well beyond the value seen in the moderately cross-resistant T-lymphoblastoid CCRF-CEM subline CCRF VCR 100. Increased mdr1/P-glycoprotein mRNA levels were also found in relapsed state acute myelogenous leukaemias (AML), and in chronic lymphocytic leukaemias (CLL) treated with chlorambucil and/or prednisone for several years. Topoisomerase I and topoisomerase II mRNA levels were found to be very variable. Whereas in all but one case of CLL topoisomerase II mRNA was not detected by slot-blot hybridizations, strong topoisomerase I and topoisomerase II gene expression levels, frequently exceeding the levels monitored in the CCRF-CEM cell line, were seen in many cell samples of acute leukaemia. If topoisomerase II mRNA was undetectable, expression of topoisomerase I was clearly visible throughout. These observations might be valuable considering the possible treatment with specific topoisomerase I or topoisomerase II inhibitors. Significant positive correlations were found (i) for topoisomerase I and histone 3.1 gene expression levels in general (P less than 0.001), and (ii) in the CLL samples additionally for the expression levels of the mdr1 gene, and the histone 3.1, topoisomerase I, and glutathione-S-transferase pi genes, respectively.

Phorbol esters induce immediate-early genes and activate cardiac gene transcription in neonatal rat myocardial cells

The mechanisms which transduce intracellular signals for the accumulation of myofibrillar protein during the onset of myocardial cell hypertrophy are unknown. Although previous studies in skeletal muscle cells have suggested that the activation of protein kinase C induces de-differentiation, including the selective disassembly of myofibrils and inhibition of myofibrillar protein synthesis, the present study demonstrates that phorbol esters which activate protein kinase C lead to the accumulation of an individual contractile protein, myosin light chain-2 (MLC-2) and produce several features of myocardial cell hypertrophy. Utilizing immunoblotting and indirect immunocytofluorescence with MLC antisera, the present study demonstrates a several-fold increase in the content of MLC-2, and a marked increase in the assembly of MLC into organized contractile units in individual neonatal rat myocardial cells following treatment with phorbol 12-myristate 13-acetate (PMA). The concentration of PMA required to elicit this response and the lack of a response with an inactive phorbol ester is consistent with the activation of a protein kinase C dependent pathway. Furthermore, PMA treatment results in the rapid induction of a program of immediate-early gene expression (including the c- fos and c- jun proto-oncogenes, and an inducible zinc finger containing gene, egr-1), and activates cardiac gene transcription as assessed by nuclear run-on analyses. The results of the present study suggest the possibility that a protein kinase C dependent pathway may be involved in the up-regulation of myofibrillar protein content and the activation of cardiac gene transcription during growth and hypertrophy of neonatal rat myocardium, and that the induction of a program of immediate-early gene expression may be linked to this response.

Selective inhibition of the canine mixed lymphocyte response by HLA-DR and DP-reactive monoclonal antibodies.

Twenty-three of 37 anti-Ia McAb reactive with human B cells, as determined by indirect immunocytofluorescence, were shown to be reactive with canine peripheral blood mononuclear cells (PBMC). Using a panel of human B cell lines that differ in their expression of HLA-DR, -DP, and -DQ molecules, it was shown that 15 of these antibodies identify HLA-DR and DP molecules (i.e., broadly reactive), while 22 identify only HLA-DR molecules. Fourteen of the 15 broadly reactive McAb were reactive with canine PBMC while only 9 of the 22 HLA-DR-specific McAb reacted with canine PBMC, suggesting that broadly reactive anti-Ia McAb are much more likely to react with canine cells than narrowly reactive McAb. Ten of the canine reactive McAb that were shown to identify typical Ia bimolecular structures on canine cells using immune precipitation analysis were tested for blocking activity in the canine mixed lymphocyte culture (MLC). All four of the broadly reactive McAb (B1F6, J-70, 9-49, and HB10a) plus two of the six narrowly reactive McAbs (H81.98.21 and H40.164.3) blocked the canine MLC when added to culture wells on day 0, suggesting that inhibition may be related to the specificity of the anti-Ia McAb employed. Since the MLC may reflect cellular interactions occurring during graft-versus-host disease, this assay may be useful for screening functionally relevant broadly reactive McAb in experimental canine bone marrow transplantation studies. These data suggest that the dog may be a useful model to study anti-Ia immunotherapy.

Monoclonal antibodies to bovine platelet factor 4: Species interactions to platelets and megakaryocytes using indirect immunocytofluorescence

Murine monoclonal antibodies (mAb) were raised to a purified product of bovine PF-4, a 9,500 dalton protein with heparin neutralization activity comparable to that of human PF-4. Using a non-radioactive slide immunoenzymatic assay, four major classes of mAb could be identified when comparisons were made between purified antigens of PF-4 and β-TG-like protein from both bovine and human species. Type 1 cross-reacted with all four antigens; type 2 reacted with PF-4s; type 3 reacted with only bovine PF-4 and β-TG-like protein; and type 4 reacted only with bovine PF-4. Differences in immunoreactivities of types 1, 2 and 3 were retained throughout the growth of succeeding clones and in ascitic fluids. Using a modified factor Xa, S-2222 chromogenic substrate-heparin inhibition assay, no mAb was found to block PF-4's ability to neutralize heparin. mAbs representative of types 1, 2 and 3 were successfully raised in stable cell lines from at least second generation clones. These were purified with protein A agarose and found to be IgG 1. By indirect immunocytofluorescence a purified type 2 mAb, 2E7, was found to specifically stain granules of human platelets and megakaryocytes, as well as masses (putative platelets within late stage megakaryocytes) without staining other cellular types in either bone marrow or peripheral blood. Species comparisons displayed positive staining for human, rat, and rabbit platelets and megakaryocytes, and negative staining for mouse, guinea pig and dog platelets and megakaryocytes. It seems likely that mAb, 2E7, is directed against an epitope, common to PF-4 of bovine, human, rabbit and rat.

Bimodal distribution of the prostaglandin I2 synthase antigen in smooth muscle cells.

Vascular and nonvascular smooth muscles in 12 different organs from the gastrointestinal, respiratory, and urogenital tracts of the rabbit, cow, dog, sheep, pig, and rat were examined for prostaglandin I2 (PGI2) synthase immunoreactivity by indirect immunocytofluorescence using monoclonal antibodies against the enzyme. Each of 35 different smooth muscle layers tested stained for the PGI2 synthase antigen except the circular smooth muscle of the rabbit large intestine. Interestingly, PGI2 synthase-positive fluorescent staining of smooth muscle was always observed to be associated with both the nuclear and plasma membranes. Our results indicate that most smooth muscle, both vascular and nonvascular, has the capacity to synthesize PGI2. Moreover, the fact that the PGI2 synthase antigen is associated with at least two different organelles in each cell suggests that there are two independent PGI2-synthesizing systems in smooth muscle; one on the nuclear membrane, and one on the plasma membrane. PGI2 formed at these different sites may subserve different functions within the same cell.

Immunocytochemical localization of the prostaglandin-forming cyclooxygenase in the cerebellar cortex

An indirect immunocytofluorescence technique was used to examine the distribution of the prostaglandin-forming cyclooxygenase in the cerebellar cortex of the pig, guinea pig, rat, mouse, cow, rabbit and sheep. Cyclooxygenase antigenicity was detected (a) in the cell bodies of Bergman glial cells in the Purkinje cell layer of the porcine, ovine and bovine cerebellar cortex; (b) in small arterioles throughout the cerebellar cortex in the sheep and cow; and (c) in the endothelial cells of large arteries in all the species examined. No cyclooxygenase-positive staining was apparent in neuronal cell bodies of granule, basket, stellate or Purkinje cells. Our results establish that prostaglandin endoperoxides can be synthesized by the arterial vasculature and at least certain glial cells in the central nervous system.

Chum salmon pituitary fractions: Somatotropic activity and cytoimmunofluorescence studies

Highly purified chum salmon pituitary fractions were bioassayed for their ability to stimulate elongation in hypophysectomized rainbow trout, Salmo gairdneri. One fraction, previously identified as the Na-retaining principle, prolactin, was not active. Of the active fractions, only the most potent one elicited a dose response. An antiserum against this fraction permitted the specific labeling of somatotropic cells in pituitary sections by an indirect immunocytofluorescence procedure. An antiserum against the Na-retaining principle led to the specific fluorescence of the follicular “prolactin cells.” However, neither antiserum appeared to discriminate between acidophile type in pituitary sections from frogs or rats.