Immpact dab substrate kit

IHC was conducted to evaluate the protein levels of TGFBR2 and FOXO3. Surgically collected pieces of kidney tissue were fixed in 4% paraformaldehyde and then embedded in paraffin. The tissues were sectioned at 4 µm thick, deparaffinized, and rehydrated. After antigen activation and blocking, kidney sections were incubated overnight at 4 °C with a TGFBR2-specific primary antibody (Fitzgerald Industries International, Acton, MA, USA) and FOXO3-specific primary antibody (Bethyl, Waltham, MA, USA). After washing, they were incubated with peroxidase-conjugated secondary antibodies for 1 h at room temperature (NICHIREI, Tokyo, Japan). Then, kidney sections were incubated with ImmPACT DAB Substrate Kit (Vector, Newark, CA, USA) for 10 min at room temperature, incubated in Meyer hematoxylin solution (Muto Pure Chemicals) for 10 s, washed, and dehydrated. Kidney sections were observed using a BZ-X710 (Keyence). The IHC positive area (brown stain) was quantified. Six images from random fields were obtained at 200× magnification. The brown stained area was measured using Keyence Hybrid Cell Count on a BZ-X Analyzer (version 1.4.1.1, Keyence). For randomization, digital images of kidney sections were segmented, numbered in sequence, and selected using a random number generator [https://www.random.org/ (accessed on 15 October 2022)].

E10.5 embryos were fixed in 4% paraformaldehyde in PBS at 4°C, rinsed in PBS, then permeabilized in PBS with 0.5% Triton X-100 for 24 h at 4°C. Neurofilament immunodetection was performed as previously described (Ray et al., 2020 (link)). Primary anti-neurofilament antibody (Developmental Studies Hybridoma Bank, 2H3) was used at a 1:20 dilution, and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch, 115-035-003) was used at 1:1000 dilution. Signal was developed using an ImmPACT DAB Substrate Kit (Vector Laboratories, SK-4105). Photographs were taken using a Nikon SMZ-U dissecting scope fitted with a Jenoptik ProgRes C5 camera.

Paraffin-embedded tissues were cut into 6-µm sections, deparaffinised in xylene, and rehydrated in phosphate buffered saline (PBS). Deparaffinised sections were treated with endogenous peroxidase blocking solution (horse serum diluted 1:1 in buffer: PBS + bovine serum albumin 1%) for 15 min at room temperature. Sections were then incubated overnight at 4 °C in rat monoclonal antibodies (mAb) specific for CD4 (clone: D7D2Z; catalogue number: #25229; Cell Signalling Technology, Tokyo, Japan; dilution 1:300) and CD8 (clone: D4W2Z; catalogue number: #98941; Cell Signalling Technology, Tokyo, Japan; dilution 1:1000). Sections were then washed in PBS buffer and biotin-conjugated secondary antibodies were then applied followed by incubation with avidin-biotin complex (Vector Laboratories: VECTASTAIN Elite ABC Kit #PK-6101) for 30 min at room temperature followed by three washes with PBS for 15 min each. Peroxidase activity was observed using an ImmPACT DAB Substrate Kit (Vector Laboratories: #SK-4105) and samples were counterstained with haematoxylin. For negative control, primary antibody was not added to the sections.