Borg decloaker rtu solution

Tissues were fixed in 10% formalin overnight, paraffin embedded, and sectioned. Deparaffinized sections were subjected to antigen retrieval using Borg Decloaker RTU solution (Biocare Medical) in a pressure cooker (Instant Pot) for 20 minutes. Sections were incubated with rabbit anti-Ki-67 (D3B5) primary antibody (1:400, CST 12202S) overnight at 4°C. Secondary antibody used was biotin-conjugated donkey anti-rabbit (Jackson ImmunoResearch), followed by detection using the Vectastain Elite ABC immunoperoxidase detection kit (Vector Labs) and Dako Liquid DAB+ Substrate (Dako). Sections were counterstained with hematoxylin and mounted with Cytoseal XYL (Thermo) for visualization. Antibody dilutions were made in Common Antibody Diluent (BioGenex). Number of crypts with >10 Ki-67 positive cells were averaged across 0.5 cm of tissue.

For histology, tissues were fixed in 10% formalin in PBS, embedded in paraffin, sectioned and stained with Hematoxylin and Eosin (H&E) or Gomori Methenamine Silver (GMS). Immunohistochemistry was performed with antibodies directed against Myeloperoxidase (MPO) to detect neutrophils or CD68 to detect macrophages. For MPO staining, antigen retrieval was performed in Biocare's Pressure Cookers using EDTA, pH 8 solution, a polyclonal rabbit anti-human myeloperoxidase primary antibody (Dako) was diluted to 1∶3500 and a Dako's Rabbit Envision detection kit was used. For CD68 staining antigen retrieval was performed with Borg Decloaker RTU solution (Biocare Medical) in a pressurized Decloaking Chamber (Biocare Medical) for 3 min. As a primary antibody rat monoclonal anti-CD68 [FA-11] antibody (Abcam) was diluted to 1∶500 followed by biotin-conjugated secondary donkey anti-rat antibodies (Jackson ImmunoResearch). The Vectastain Elite ABC immunoperoxidase detection kit (Vector Labs PK-6101) followed by Dako Liquid DAB1 Substrate (Dako) was used for visualization. For quantification of neutrophil and macrophage numbers, 5×100 grids were counted at a 40x magnification for 3 individual animals.

Mouse small intestines and colons were harvested and fixed in 4% formalin, embedded in paraffin, and sectioned for staining with Hematoxylin–Eosin (H&E) or Alcian blue-periodic Acid-Shiff (AB/PAS) solutions. For immunohistochemistry, Borg Decloaker RTU solution (Biocare Medical) was used for antigen retrieval in a pressurized Decloaking Chamber (Biocare Medical) for 5 min. Antibodies used: rabbit anti-Ki-67 (1:4000, Cell Signaling 122,025), rat anti-BrdU (1:2000, Abcam 6326), rabbit polyclonal anti-RFP (1:500, Rockland 600–401-379), biotin-conjugated secondary donkey anti-rabbit or anti-rat (Jackson ImmunoResearch). For visualization, Vectastain Elite ABC immunoperoxidase detection kit (Vector Labs PK-6101) and Dako Liquid DAB + Substrate (Dako) were used. All antibody incubations were performed with Common SignalStain (R) Antibody Diluent (Cell Signaling 8112L). For EdU histological analysis, Click-iT reaction was performed on paraffin slides following the manufacturer's protocol (Thermo Fisher). For all histological analysis, individual crypts were analyzed, with an average of 150 crypts per mouse.