Journal of Histochemistry & Cytochemistry最新文献

We tracked prosaposin (PSAP), a trophic factor, using an antibody specific to its proteolytic portion and an antibody to sortilin that traffics PSAP only to the lysosome. Immunostaining revealed that PSAP was distributed mainly on the basal side of seminiferous tubules, where many Sertoli cells and pachytene spermatocytes contained PSAP and its distribution differed depending on the stage of the spermatogenic cycle. The PSAP-sortilin complex was sorted to large lysosomes in the basal cytoplasm of Sertoli cells, where it may be processed into saposins. In contrast, in the thinner apical cytoplasm of Sertoli cells, PSAP in small lysosomes was transported to the apical side around sperm heads or into the lumen for secretion. The results of in situ hybridization analyses suggested that immature tubular cells in young animals produce PSAP to self-stimulate proliferation. However, in adults, not only Sertoli cells but also pachytene spermatocytes produce and secrete PSAP around germ cells or into the tubular lumen to stimulate cell proliferation or differentiation in a paracrine or autocrine manner. In summary, PSAP is not only a precursor of lysosomal enzymes but also a pivotal trophic factor in organogenesis in the immature testis and spermatogenesis in the mature testis.

Gelatin zymography is widely used to detect gelatinase activity, which is performed on unfixed tissue because it is assumed that fixation inactivates enzymes. However, using fixed tissues has several advantages over using fresh tissues for such prevention of tissue decay, thereby preserving the proteins as well as the morphology and structure of the specimens. In this study, we investigated the effects of the four commonly used fixatives (ethanol, acetone, zinc-based fixative (ZBF), and paraformaldehyde (PFA)) on the gelatinolytic activity in mouse brain tissue. Multiple protocols were employed to extract proteins from the fixed brain tissue. Western blotting and in-gel zymography (IGZ) were used to detect the gelatinase proteins and gelatinolytic activity of the extractions, respectively. In situ zymography (ISZ) revealed that ethanol, acetone, ZBF, and short-time PFA fixation did not inhibit gelatinolytic activity. Neither 1% Triton + 1 M NaCl nor 10% DMSO + 1 M NaCl was effective in extracting proteins from ethanol-, acetone-, ZBF-, or PFA-fixed brain tissues. However, 8 M urea + 4% CHAPS effectively extracted gelatinase proteins from ethanol- and acetone-fixed tissues while retaining the gelatinolytic activity. 2% SDS effectively extracted gelatinase proteins from ethanol-, acetone-, and ZBF-fixed tissues while retaining the gelatinolytic activity. Although 2% SDS + heating extracted gelatinase proteins from ethanol-, acetone-, ZBF-, and even long-term PFA-fixed tissues, the gelatinolytic activity was not retained. Our findings suggest that both ISZ and IGZ can be performed on fixed brain tissue, which is anticipated to be an improvement over the conventionally used gelatin zymography methods. (J Histochem Cytochem 71: 481-493, 2023).

The author of the accompanying classic paper from the Journal of Histochemistry and Cytochemistry (Crouch JJ, Sakaguchi N, Lytle C, Schulte BA. Immunohistochemical localization of the Na-K-Cl Co-transporter (NKCC1) in the Gerbil Inner Ear. Journal of Histochemistry & Cytochemistry 1997;45(6):773-778) comments on how the immunohistochemical techniques used in the study provided critical new information that helped define the cellular and molecular mechanisms involved in the generation and maintenance of electrochemical gradients in the ear, particularly the presence of the Na,K,2Cl symporter (NKCC) in the inner ear. (J Histochem Cytochem 71: 509-510, 2023).

I wanted to be a cytochemist but encountered detours and then, in some of my work, became one of a different kind than classically defined. I recount this here to discourage young scientists from regarding cytochemistry as something that peaked in the past, but rather to be viewed as an entirely new form of the discipline, and so rich with opportunities. (J Histochem Cytochem 71: 475-480, 2023).

Recently there have been reports that identify two transient receptor potential channels in cell-matrix junctions known as focal adhesions. These are the calcium channel TRP canonical 7 and the calcium-activated monovalent ion channel, TRP melastatin (TRPM) 4. Here, we report on the occurrence of TRPM4 in focal adhesions of fibroblasts. Of three commercial antibodies recognizing this channel, only one yielded focal adhesion staining, while the other two did not. The epitope recognized by the focal adhesion-localizing antibody was mapped to the extreme C-terminus of the TRPM4 protein. The other two antibodies bind to N-terminal regions of the TRPM4 proteins. Deletion of the TRPM4 gene by CRISPR/cas9 techniques confirmed that this channel is a bona fide focal adhesion component, while expression of full-length TRPM4 proteins suggested that processing may occur to yield a form that localizes to focal adhesions. Given the reports that this channel may influence migratory behavior of cells and is linked to cardiovascular disease, TRPM4 functions in adhesion should be explored in greater depth. (J Histochem Cytochem 71: 495-508, 2023).

Telomerase reverse transcriptase (TERT) gene aberrancies correlate to adverse prognosis in follicular thyroid carcinoma (FTC). As loss of 5-hydroxymethylcytosine (5hmC) has been associated with TERT promoter mutations in papillary thyroid carcinoma, this study sought to analyze the levels of 5hmC in a cohort of follicular thyroid tumors with available TERT data. A total of 29 tumors (26 FTCs, 2 follicular thyroid tumors of uncertain malignant potential, and 1 oncocytic thyroid carcinoma) with known TERT promoter mutational status and TERT gene expression were assessed for 5hmC immunoreactivity using two antibodies (clones RM236 and 4D9.) Slides were analyzed using a semiquantitative scoring system. Of the 10 tumor cases with aberrant TERT, only 1 scored negative with both antibodies (1/10; 10%), whereas the remaining 9 cases (9/10; 90%) exhibited some positivity for at least one antibody. Of the 19 TERT wild-type tumors, no case was scored negative using RM236, and 2 cases (2/19; 11%) using 4D9. The differences between TERT promoter mutated and wild-type groups were non-significant. The sensitivity and specificity for 5hmC immunohistochemistry (IHC) to detect mutated cases were 10% and 100% (RM236) and 20% and 89% (4D9). Therefore, 5hmC IHC is not a sensitive marker for detecting TERT promoter mutations in follicular thyroid tumors.

The glycan moiety Lewis X (LeX) has been implicated in defining progenitor cells as well as playing a role in the progression of solid tumors, including breast cancer. Here, we used the original stage-specific embryonic antigen-1 (SSEA-1) antibody, MC-480, targeting the LeX motif to examine the expression pattern of this marker within the context of a differentiation hierarchy as well as functional properties of breast cancer cells. Immunohistochemical staining revealed the presence of SSEA-1 in a progenitor zone in the normal breast gland. In breast cancer, 81 of 220 carcinomas (37%) were positive for SSEA-1 and a distinct pattern could be correlated to major subtypes. Specifically, estrogen receptor alpha (ERα)-negative tumors showed a higher frequency of SSEA-1 expression compared to ERα-positive tumors, which are generally considered more differentiated (56% vs 29%, p<0.005). Functional assays performed on two representative breast cancer cell lines demonstrated that SSEA-1-expressing cells exhibited cancer stem cell properties as well as having more invasive potential, regardless of ERα status. A potential role of SSEA-1 in metastasis was confirmed by pairwise staining of primary- and corresponding lymph node tumors. Altogether, our data suggest that expression of SSEA-1 in breast cancer contributes to the malignant phenotype.

The authors of the accompanying classic paper from the Journal of Histochemistry and Cytochemistry (Evanko SP, Wight TN. Intracellular Localization of Hyaluronan in Proliferating Cells. Journal of Histochemistry & Cytochemistry. 1999;47[10]:1331-1341) comment on the impact and significance of their findings on the intracellular localization of hyaluronan in arterial smooth muscle cells using immunohistochemical techniques. These seminal findings signaled the potential for a role of hyaluronan in the functions of microtubules and mitosis.