An Intermittent CytochemistPederson, Thoru
doi: 10.1369/00221554231195393pmid: 37610161
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)
Gelatin Zymography Can Be Performed on Fixed Brain TissueChen, Zhenzhou; Chuang, Dennis; Chen, Shanyan; He, Qiwei; Tomlison, Brittany N.; Cui, Jiankun; Gu, Zezong
doi: 10.1369/00221554231194118pmid: 37599425
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)
Two Transient Receptor Potential Channels at Focal AdhesionsMitsou, Ioli; Carlson, Cathrine Rein; Multhaupt, Hinke A.B.; Brakebusch, Cord; Couchman, John R.
doi: 10.1369/00221554231194119pmid: 37596792
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)