Peroxidase dab kit

Immunohistochemistry was performed on 2 µm-thick sections obtained from formalin-fixed tissue embedded in paraffin. After dewaxing and rehydrating, heat-induced epitope retrieval was performed by boiling the slides with EDTA (pH 9) (Dako, Glostrup, Denmark). Endogenous peroxidase was blocked with 3% hydrogen peroxide followed by incubation with mouse-to-mouse blocking reagent to inhibit endogenous mouse immunoglobulin and then another blocking with blocked BSA 5%. Sections were incubated overnight at +4 °C with mouse monoclonal α-MF20 antibody (dilution 1:50; DSHB) or rabbit monoclonal α-SNAI2 (dilution 1:100). Detection of the primary antibody was performed by using the appropriate secondary biotinylated antibody (Dako, Carpinteria, USA) and the peroxidase DAB kit (Dako, Carpinteria, USA) with or without counterstaining with Gill’s hematoxylin (Dako, Carpinteria, USA). Negative controls were stained in parallel with either isotype non-specific IgG or only the primary antibody. The light microscopy imaging was performed on a Nikon E600 light microscope equipped with NIS Elements BR software, using 20x objective.

Immunohistochemistry was performed on 2 μm thick sections obtained from formalin-fixed tissue embedded in paraffin. After dewaxing and rehydrating, heat-induced epitope retrieval was performed by boiling the slides with sodium citrate (pH 6) (Dako, Glostrup, Denmark). Endogenous peroxidase was blocked with 3% hydrogen peroxide and then with 5% bovine serum albumin (BSA). Sections were incubated overnight at 4°C with rabbit monoclonal Cleaved Caspase-3 antibody (1:300) (9661S, Cell Signaling Technology, Danvers, MA, USA). Detection of the primary antibody was performed by using the appropriate secondary biotinylated antibody (K8024) (ready to use) (Dako, Carpinteria, CA, USA) and the peroxidase DAB kit (Dako, Carpinteria, CA, USA). Counterstaining was performed with hematoxylin solution Gill2.
The entire slides were scanned and captured by NanoZoomer S60 Digital scanner, 5 random 20X fields of the subcapsular cortex for each mouse were selected using the NDP.view2 Image viewing software. The mean intensity of caspase 3 staining was measured by Image J software.

The excised colon from each mouse was opened longitudinally, “swiss rolled”, and fixed for 24 h in 10% (w/v) buffered formalin at room temperature. All tissues were embedded in paraffin, and 5-μm thick sections were stained with Haematoxylin and Eosin (H&E). Unstained sections were deparaffinised in xylene and rehydrated in a graded series of ethanol (100–70% v/v) finishing in dH₂O. Antigen retrieval was achieved by boiling slides in citrate buffer (ThermoFisher Scientific, AP-9003-500) or EDTA-Tris (1 mM EDTA, 10 mM Tris pH 9) for 15 min, followed by incubation in 3% (v/v) H₂O₂ to inhibit endogenous peroxidases. Sections were blocked in 5% (v/v) goat serum and incubated with primary antibodies against Bcl-G (clone 2E11, [9 (link)]), BrdU (BD Biosciences, BD555627), Ki67 (Cell Signaling, CST 9129), Caspase-3 (Cell Signaling, CST 9664), CD45 (BD Biosciences, BD553076), or p53 (Leica Biosystems, P53-CM5P-L) overnight at 4 °C in a humidified chamber. Sections were rinsed, then incubated in HRP-conjugated biotinylated secondary antibodies for 1 h at room temperature, after which HRP (Vector Laboratories) was detected using a DAB Peroxidase kit (Dako, K346811-2). All sections were counterstained with Haematoxylin. The extent of positive staining per mm [2 (link)] of tissue was quantified using ImageJ software.

The ileum or distal colon was flushed with PBS and fixed in 10% buffered formalin. Immunofluorescence staining was performed as described previously41 (link). Briefly, rehydrated paraffin or frozen sections were blocked using 5% normal goat serum or M.O.M mouse Ig blocking reagent (Vector Lab) and treated with primary antibody at 4 °C overnight. The sections were further incubated with secondary antibodies conjugated with Alexa 488 (Life Technologies), counterstained with DAPI and visualized with a LSM 510 Meta confocal microscope (Carl Zeiss). For immunohistochemistry, re-hydrated paraffin sections were treated with 3% H₂O₂ in deionized water and immunostaining was performed using Vectastatin ABC kit (Vector Lab) according to the manufacturer’s protocols. Antigen-antibody complexes were detected with a DAB peroxidase kit (Dako). For Tff3 immunohistochemistry, a sample of the distal colon with a fecal pellet was collected and fixed in methanol-Carnoy’s solution overnight38 (link). The sources of the primary antibodies are in the Supplementary Materials.