Signalstain dab substrate

Gastric tissue samples from WT and Myd88^−/− control and H. felis-infected mice were incubated with specific antibodies overnight (Supplementary Table 2) after antigen retrieval in a pressure cooker using 0.01 M sodium citrate. These antibodies have been previously validated by our group and others^{36 (link)–38 (link)}. After incubation with HRP-conjugated secondary antibodies, sections were developed using SignalStain DAB substrate (Cell Signaling) following manufacturer’s instructions. A secondary antibody only control was included to ensure that the secondary antibody did not unspecifically bind to certain cellular compartments. Samples were counterstained with hematoxylin (Morphisto). For automated image acquisition, Olympus Virtual Slide System VS120 (Olympus) was used. Stainings were visually quantified in a blinded manner. Five random high power field (HPF) areas per sample were scored blinded.

Tissue fixation, dehydration, paraffin embedding, and hematoxylin and eosin (H&E) staining were performed as previously described^{44 (link)}. Briefly, harvested tissues were fixed in 10% formalin, dehydrated, paraffin embedded. Five-micron sections were stained with the custom αhPRLrI primary antibody (New England Peptide) at a 1:12,500 titer, following an additional 10% formalin fixation blocking step prior to antigen retrieval. After washing, slides were incubated with SignalStain Boost IHC Detection Reagent (8114 S, Cell Signaling) for 1 h, followed by incubation with SignalStain DAB Substrate (8059 S, Cell Signaling) for 1 min. Slides were counterstained with hematoxylin (Gill No. 3; GHS332, Sigma), dehydrated, and coverslipped using Permount mounting medium (50–277–98, Electron Microscopy Sciences). hPRLrI staining was expressed as an Allred score, namely the sum of the proportion of positive cells (0–5 scale) and mean intensity (0–3 scale)^{44 (link)}.

Formalin-fixed paraffin-embedded kidney specimens that were obtained from Wistar control and Wistar STZ rats were deparaffined with Histochoice Cleaning Agent (Sigma Aldrich) and ethanol and hydrated in water. Epitopes were retrieved by incubating the specimens in sodium citrate buffer (10 mM, pH 6.0) at 97 °C for 20 min. The specimens were blocked with 5% bovine serum albumin (BSA) for 1 h. Bovine serum albumin was then removed from the slides, and primary antibodies (diluted in 5% BSA) were added to the specimens, followed by overnight incubation at 4 °C (Table 2). The next day, the specimens were incubated with secondary antibodies (Cell Signaling Technology, Danvers, MA, USA, catalog no. 8114S or 8125S) at room temperature for 30 min. The slides were then incubated for 2 min with Signal Stain DAB substrate (Cell Signaling Technology). The specimens were counterstained with hematoxylin for 5 s and dehydrated with ethanol and Histochoice. Cells were imaged using a Nikon Eclipse Ti microscope. Quantification of the amount of stained proteins was performed using ImageJ 1.52a software (National Institutes of Health).