Impress reagent kit

Whole-mounts of MGs of pubertal, adult virgin, or lactation day-0 mice were fixed and
processed as previously described (Jones et al.,
1996). For histology and immunohistochemical staining, MGs were fixed in
10% formalin and embedded in paraffin. For RUNX1 or ERα detection, antigen
retrieval (Citrate buffer pH 6.0, 20 min boil in microwave oven) was performed prior
to incubation with an anti-RUNX1 antibody (2593-1, Epitomics, Burlingame, CA) or an
anti-ERα antibody (SC-542, Santa Cruz Biotechnology, Dallas, TX). Signal was
detected using the impress reagent kit and DAB substrate (MP-7401 and SK-4100, Vector
Laboratories, Burlingame, CA).

For IHC studies, section(s) were taken on poly-L-lysine coated slides and then subjected to a clearing which was followed by rehydration. The antigen retrieval was carried out in citrate buffer using EZ-Retriever^® (Biogenex, USA). These slides were then washed in phosphate buffer saline for 20 min. Serum blocking was done using normal horse serum, and subsequently, non-specific binding and endogenous peroxidase blocking were followed by overnight incubation with polyclonal antibody for P. multocida that was standardized at the dilution of 1:2500. It was then followed by 20 min incubation with secondary antibody (Vector, impress reagent kit; peroxidase universal antimouse/rabbit Ig). Color was developed with substrate diaminobenzidine (vector) and counterstained with Gill’s hematoxylin (Merck, Germany) stain. Omission of primary antibodies was used for negative control. Further, IHC scoring was done on the basis of antigen positive cells and classified as mild, moderate, and severe.

For antibody concentration optimisation, we obtained tissue slides from the tissue science facility. For this, the tissue sections (4 µm) were cut and mounted on super-frost, positively charged glass slides. Different antibody concentration was used to stain the tissues. The universal secondary antibody from Impress reagent kit (VECTOR, Burlingame, CA) were employed for mouse/rabbit generated antibody. For goat antibodies, anti-goat HRP was used. After incubation with the primary and secondary antibody and subsequent washing, the colour was developed with the peroxidase kit (VECTOR).