Ihc protein block solution

Tissue samples were fixed with 10% formaldehyde, embedded in paraffin, and cut into 4-μm-thick sections. Sections were deparaffinized in xylene and then rehydrated by alcohol series. To examine the histology, sections were stained with hematoxylin and eosin (H&E). For immunohistochemistry, sections were first treated with 3% H₂O₂ to block the endogenous peroxidase, then incubated with IHC protein block solution (DAKO, Carpinteria, CA). Sections were then reacted with primary antibody at 4°C for overnight, then sequentially reacted with horseradish peroxidase-conjugated secondary antibody (DAKO). After washing, sections were incubated with diaminobenzidine tetrachloride solution and counterstained with Mayer’s hematoxylin. For double immunofluorescence staining, sections were incubated with primary antibodies, then incubated with fluorescence-conjugated secondary antibodies (Abcam, Cambridge, UK). Immunofluorescence signal was detected under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The following primary antibodies were used: Crif1 (Santa Cruz Biotechnology, Santa Cruz, CA), MTCO1 (Abcam), K15 (Abcam), K5 (Santa Cruz Biotechnology, Santa Cruz, CA), AE15 (Thermo Fisher Scientific), AE13 (Abcam), Lgr5 (Thermo Fisher Scientific) and Ki67 (Vector Laboratories, Burlingame, CA).

Tissue samples were fixed with 10% formaldehyde, embedded in paraffin, and cut into 4 μm thick sections. Sections were deparaffinized in xylene and then rehydrated using an alcohol series. For IHC, sections were first treated with 3% H₂O₂ to block the endogenous peroxidase and then incubated with IHC protein block solution (DAKO, Carpinteria, CA, USA). The sections were then reacted with light chain 3 (LC3) antibody (Sigma-Aldrich, St. Louis, MO, USA) at 4°C for overnight, followed by horseradish peroxidase-conjugated secondary antibodies (DAKO). After washing, the sections were incubated with diaminobenzidine tetrachloride solution and counterstained with Mayer's hematoxylin.

For the immunohistochemical analysis, the skin tissues were fixed using a solution of 10% formaldehyde (v/v). Subsequently, these tissues were embedded in paraffin, sectioned into slices measuring 4 μM-thick sections, deparaffinized with xylene, and then rehydrated gradually with a series of alcohol baths. In preparation for the immunohistochemical staining procedure, the sections underwent treatment with 3% (v/v) H₂O₂ solution to block endogenous peroxidase. Following this, the sections were gently incubated with an IHC protein block solution (DAKO, Carpinteria, CA, USA, S2002). Once properly conditioned, the sections were subjected to an overnight incubation at a temperature of 4 °C with a primary WWOX antibody (Abcam, Cambridge, UK, ab238144). Subsequently, they were then incubated with horseradish peroxidase-conjugated secondary antibodies (DAKO, K4003) for 2 h at room temperature. After washing, the sections were exposed to a solution of diaminobenzidine tetrahydrochloride and counterstained with Mayer’s hematoxylin. Quantitative analysis of immunohistochemical staining was performed using ImageJ software v1.53a (WS Rasband, National Institutes of Health, Bethesda, MD, USA).