Bcip nbt

To quantify LGR5+ cells in jejunal tissues, formalin-fixed tissue sections were stained with anti- LGR5 antibodies using the Mach3 Rabbit AP-Polymer Detection Kit (Biocare Medical, USA) as performed earlier (31 (link)). The paraffin-embedded tissue sections were deparaffinized and epitope retrieved by heating the tissue sections in low pH citrate buffer (Vector Laboratories, USA) using a microwave. After blocking with a serum-free protein blocker (Agilent Dako, USA) for 30 min, the tissue sections were incubated with anti-LGR5 primary antibodies (Supplementary Table 1). The negative control slide consisted of rabbit Ig fractions (R&D Systems, USA) used at the same isotype and concentration as LGR5 antibodies to determine the background intensity and staining. The tissue sections were subsequently treated with the kit’s probe and polymer and finally developed using BCIP/NBT (Agilent Dako) chromogen system. The slides were then mounted with Vecta Mount AQ (Vector Laboratories). An average of 19–20 view fields (0.116mm²/view field under a 400 x magnification) were enumerated in each of the slides to quantify LGR5+ cells manually. The tissue sites for this evaluation were selected randomly. Tissues were analyzed by two different blinded individuals to avoid bias and averaged prior to the statistical analysis.

To quantify TGF-β⁺ cells in jejunal tissues, formalin-fixed tissue sections were stained with anti-TGF-β antibodies using the Mach3 Mouse AP-Polymer detection kit (Biocare Medical, Pacheco, CA, USA) as done previously [1 (link)]. The paraffin-embedded tissue sections were deparaffinized and epitope retrieved by heating the tissue sections in citrate buffer (Vector Laboratories, Burlingame, CA, USA) in a microwave. After blocking with a serum-free protein blocker (Vector Laboratories) for 30 min, the tissue sections were incubated with anti-TGF-β1 primary antibodies (Supplemental Table S1). The negative control slide consisted of mouse Ig fractions (Agilent Dako, Santa Clara, CA, USA) used at the same isotype and concentration as TGF-β antibodies to determine the background intensity and staining. The tissue sections were then treated with the kit’s probe and polymer and finally developed using the BCIP/NBT (Agilent Dako) chromogen system. The slides were then mounted with Vecta Mount AQ (Vector Laboratories). An average of 19–20 view fields (40× magnification, view field area at 40× magnification was 0.116 mm²) were used in each of the slides to quantify TGF-β⁺ cells manually using SPOT3 live imaging software. The tissue sites for this evaluation were selected randomly and counted by two different individuals to avoid bias.

For visualization of apoptosis and viral antigen colocalization, a sequential IHC protocol was followed. First, the apoptosis assay (HRP) was performed as described above. Modifications for this described assay included an additional blocking step with Dual Endogenous Block (Dako) following the hydrogen peroxidase incubation. Following incubation with the Vector NovaRed chromogen, slides were washed in ddH20. The slides were then incubated with an in-house generated rabbit polyclonal anti-VARV hyperimmune sera antibody (1 : 5000). The secondary antibody that was then utilized was a swine anti-rabbit alkaline phosphatase purified antibody (Dako). BCIP/NBT (Dako) was then used as the chromogen to create a blue-black color in the presence of viral antigen. Counterstaining for this double-stain IHC assay was done with a nuclei methyl green stain. Slides from PBS inoculated prairie dogs were used as negative controls.