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).
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