Publication protocol
IHC was conducted with sections mounted on poly-L-lysine-coated slides using a modified biotin-peroxidase complex method as previously described (35). Briefly, tissue sections were incubated overnight at 4°C with rabbit polyclonal antibodies against IRE1α (dilution, 1:150; cat. no. sc-20790; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), IRE1β (dilution, 1:100; cat. no. GTX87426; GeneTex, Inc., Irvine, CA, USA) and MUC2 (dilution, 1:100; cat. no. ab76774; Abcam, Cambridge, UK). Antigen-antibody complexes were detected with a biotinylated goat anti-rabbit antibody (dilution, 1:300; cat. no. SA1020; Boster Biological Technology Co., Ltd., Wuhan, China) conjugated with streptavidin-horseradish peroxidase (HRP) for 30 min at 37°C. At room temperature and visualized by reacting with the 3,3-diaminobenzidine reagent kit (Solarbio Science & Technology Co., Ltd., Beijing, China; cat. no. DA1010) at room temperature for 5 min. Sections were counterstained with 1% hematoxylin for 1–10 min at room temperature. Negative control sections were obtained by omitting the primary antibody or by using an unspecific antibody. Images at a magnification of ×400 were captured using a Nikon Ds-Fi2 500w light microscope, (Nikon Corporation, Tokyo, Japan). IHC staining was used to semi-quantitatively determine protein levels as previously described (35): A total of 10 fields were randomly selected on each slide and 100 cells per field were counted and scored for IRE1α, IRE1β and MUC2 staining. The semi-quantitative scores were obtained from the staining intensity (none, 0; weak, 1; moderate, 2; strong, 3) multiplied by the percentage of positively stained cells (≤5%, 0; 6–25%, 1; 26–50%, 2; 51–75%, 3; and >75%, 4).
Full paper
Login or
join for free to view the full paper.
Videos
Check out videos that might be relevant for performing Immunohistochemistry Mouse - Muc-2 using Anti-MUC2 antibody (ab76774) from Abcam. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.
We haven't found any additional videos for this experiment / product combination yet.