Hrp conjugated anti mouse secondary antibody

Cerebella were isolated and homogenized in buffer containing 0.32 M sucrose, 10 mM Tris–HCl (pH 7.4), 1 mM EDTA and protease inhibitors (cOmplete Mini, EDTA free, Roche, Mannheim, Germany). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P membrane (Millipore, Billerica, MA, USA). The membrane was incubated with anti-mGluR5 antibody (1:5000, ab76316, Abcam, Cambridge, UK) and β-actin (1:1000, AC-74, Sigma-Aldrich, St. Louis, MO, USA) followed by anti-rabbit HRP-conjugated secondary antibody (1:7000, Jackson ImmunoResearch, West Grove, PA, USA) or anti-mouse HRP-conjugated secondary antibody (1:7000, Jackson ImmunoResearch). The signals were visualized by ECL Prime detection reagents (RPN2232, Cytiva, Marlborough, MA, USA).

Rats’ pancreases were fixed in aqueous Bouin solution for 24 h and embedded in paraffin (Labonord, Templemars, France). The entire pancreas was homogeneously sampled using a fixed-interval sampling method to avoid bias due to regional change in islets distribution, as previously described [24 (link)]. Briefly, each paraffin block was serially sectioned (5 μm) throughout its full-length, sections at a fixed interval were mounted on slides and stored at room temperature for future immune-staining.
Pancreatic sections were blocked for 1 h with 10% normal goat serum in Tris buffer saline and then probed for insulin using primary mouse anti-insulin antibody (Santa Cruz, France) (1:200). Sections were then incubated with an HRP conjugated anti-mouse secondary antibody (Jackson immunoResearch Laboratories, Ely, UK) (1:200). β cells areas were determined by morphometric analysis in pancreatic sections stained for insulin, using an OLYMPUS BX60 microscope equipped with Histolab 10.5.1 computer-assisted image analysis system software (Microvision Instrument, Evry, France). The relative β cell area in each stained section was calculated as the ratio of insulin positive cells area over the total pancreatic cells area [24 (link),25 (link)]. At least six sections homogeneously distributed were analyzed for each pancreas.

Enzyme-linked immunosorbent assay (ELISA) kits for thyroid hormone assays were purchased from Life Technologies Pvt. Ltd., India. For estimations of total cholesterol, triglyceride and high density lipoprotein cholesterol, assay kits were obtained from Span diagnostics Pvt. Ltd., Surat, India. Kits for aminotransferase enzymes, alanine transaminase, aspartate transaminase, and lactate dehydrogenase were from Erba diagnostic pvt. Ltd., GmbH, Germany. ELISA kit for TNF-α was purchased from Ray Biotech Inc, Norcross, GA, USA. While anti-TPO, anti-TSHR, anti-β-actin and nitrocellulose were obtained from Santa Cruz Biotechnology, USA; NC membrane was supplied by Millipore, USA. Jackson Immuno-Research Laboratories Inc. USA supplied HRP-conjugated anti-mouse secondary antibody. Bovine serum albumin (BSA) and Tween-20 were provided by Sigma-Aldrich, USA. All other routine chemicals used in the biochemical studies were purchased from Hi-Media, Mumbai, India.

Cell pellets were resuspended in 200 μl lysis buffer (8 M urea, 50 mM Tris–pH 7.5, protease inhibitors (Merck)). From there on, processing, SDS–PAGE separation, Western blotting and fluorescent‐based imaging was done as described in Eisenberg‐Bord et al (2021 (link)), with the exception of the HA blot in Fig EV1C. Here, the SDS–PAGE gel was blotted onto PVDF membrane (Millipore) by wet transfer, and imaging was done using X‐ray film (FujiFilm) to detect signal from HRP‐conjugated anti‐mouse secondary antibody (1:7,500, Jackson ImmunoResearch, #111‐035‐003) incubated with ECL substrate (Thermo Scientific). The following antibodies were used for Western blot: anti‐HA (1:1,000, BioLegend, #901502), anti‐Myc (1:3,000, Abcam, #ab9106), anti‐Histone H3 (1:5,000, Abcam, #ab1791), anti‐Sec61 (1:5,000, a kind gift from Matthias Seedorf of Heidelberg University and Marius Lemberg of the University of Cologne), anti‐Actin (1:2,000, Abcam, #ab8224), goat anti‐rabbit IgG H&L 800CW (1:7,500, Abcam, #ab216773) and goat anti‐mouse IgG H&L 680RD (1:7,500, Abcam, #ab216776). Membranes were incubated for 1 h at RT with fluorescent streptavidin (1:10,000, Invitrogen, #S11378) diluted in 2% (w/v) BSA/PBS containing 0.01% NaN₃ to detect biotinylated proteins.