K4003

The terminal part of the small intestinal tissue was harvested, fixed in formalin (#133-10311; FUJIFILM Wako), and embedded in paraffin. Using the sections of hematoxylin–eosin staining, three intact crypts were randomly selected from each sample, and Paneth cells were manually counted under a microscope (400× magnification) by a researcher who was blinded to the mouse genotypes. For immunohistochemistry, the following primary antibodies were used: Ki67 (1:100; #12202; Cell Signaling) and LAT1 (provided by Osaka University [19 (link)]). The secondary antibody #K4003(Dako) was used for Ki67 staining. For LAT1 staining, the ABCHRP Kit Peroxidase (#pk6101; Vector Laboratories) was used. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for the analysis of Ki67-positive cells by two researchers who were blinded to the mouse genotypes.

Tissues were fixed in formalin for 24 hours and paraffin-embedded. Four micron sections were deparaffinized and antigen retrieval was performed with either citrate buffer, pH 6 (for Ki67) or Tris-EDTA buffer, pH 9 (for caspase-3, CD3 and CD31). Sections were boiled in retrieval buffer for 20 minutes, then cooled for 20 minutes. Using an automated slide stainer (Dako Australia, North Sydney, NSW, Australia) sections were quenched with hydrogen peroxide, incubated with primary antibody then secondary antibody (K4003, Dako) and revealed with diaminobenzidine (Dako) or ImmPact NovaRed Peroxidase Substrate (Vector Laboratories, Burlingame, CA). Sections were counterstained in Mayer's hematoxylin. Primary antibodies were: Ki67 (ab66155, Abcam, final dilution 1:250), cleaved caspase-3 (CST9661, Cell Signaling Technology, Beverly, MA, final dilution 1:300), CD3 (A0452, Dako, final dilution 1:400) and CD31 (ab28364, Abcam, final dilution 1:100). Images were analyzed using CellProfiler, Broad Institute, MA [59 (link)], quantifying the number of positively stained pixels or total number of cells, identified by hematoxylin staining and the number of diaminobenzidine- or ImmPact NovaRed-positive cells present in the images. See Supplementary Methods for further details.

The paraffin-free tissue sections were treated with citrate antigen repair buffer (pH 6.0), and then the tissue sections were incubated with peroxidase blocking solution (S2023, Dako) for 15 min and blocked with protein (X0909, Dako) for 30 min. The corresponding specific primary antibodies were used: phosphor-ser221-MEK (rabbit, ab96379, Abcam), phospho-ERK1/2-Thr202/Tyr204 (rabbit, 4370, CST) and phospho-ser473-AKT (rabbit, 4060, CST). After treatment, the slides were incubated overnight at 4°C. Rabbit HRP conjugated secondary antibody (K4003, Dako) and hematoxylin (MHS32, Sigma) counterstain were used for treatment. The samples were observed with a digital microscope (Panoramic Viewer, 3D HISTECH), and protein expression was measured with a histochemical score (H-Score). Immunohistochemical results were scored with H-SCORE. The number of positive cells in each section H-SCORE = ∑ (PI × I) = (percentage of weak-strength cells × 1) + (percentage of medium-strength cells × 2) + percentage of cells. In this formula, PI represents the percentage of positive cells in the section, and I represents the staining intensity (9 –11 (link)).

Serially cut frozen slides were air-dried and fixed in prechilled 100% acetone). Slides were rinsed in distilled water and then washed in TBS-T. Endogenous peroxidase activity was quenched via incubation with 5% (v/v) H₂O₂ (Sigma-Aldrich, H3410) in dH₂O. After 30 minutes, the slides were washed and incubated in serum-free protein block (DAKO, X0909) for 30 minutes, after which excess liquid was removed. Serial sections were then incubated with 1:1000 (v/v) anti-MPO (mouse, monoclonal; Abcam, ab25989) or 1:1000 (v/v) anti-NE (rabbit, monoclonal; Abcam, ab131260) or 1:500 (v/v) anti-CitH3 (rabbit, monoclonal; Abcam, ab219407) for 1 hour in antibody diluent (bovine serum albumin [1% w/v], Sigma-Aldrich, A7906) and Triton-X100 (0.5% v/v, Sigma-Aldrich, 270733) in TBS-T. Slides were further washed in TBS-T and incubated with horse radish peroxidase (HRP)-coupled secondary antibody (anti-mouse, DAKO, K4001; anti-rabbit, K4003) for 30 minutes. Following washing in TBS-T, slides were incubated for 10 minutes with Opal690 fluorophore (PerkinElmer, FP1497) in tyramide signal amplification (TSA) buffer (PerkinElmer, FP1498, 1:50 v/v dilution in 1x plus amplification buffer) and subsequently washed. Specimens were incubated in a 1:800 (v/v) dilution of Spectral DAPI for 10 minutes, washed, and then cover-slipped with fluorescence mounting medium.

DSCAM expression pattern was assessed in a patient by comparing the aganglionic and ganglionic segments, together with non-HSCR colon tissue from patient undergoing colostomy through immunostaining. Colon samples from the HSCR patients and patient for colostomy in this study were obtained at the time of the surgery. Immunohistochemical staining for DSCAM was performed using 4 μm deparaffinized sections. The slides were prepared following standard procedures and stained with an anti-DSCAM primary antibody (Atlas Antibodies, HPA019324, 1:1000) and secondary antibody (anti-rabbit (DAKO, K4003)).

Fixed kidney samples were embedded in paraffin and sectioned. Kidney structure of the offspring was examined using hematoxylin and eosin (H&E) and periodic acid Schiff stain (PAS).
For IHC staining, kidney sections were incubated with primary antibodies against FGF2, GDNF (Santa Cruz Biotechnology, CA, USA) and Pax 2 (Abcam, Cambridge, UK). The tissues were then incubated with polymer secondary anti-rabbit antibodies (Dako Ref K4003) and horseradish peroxidise enzyme and DAB+ (liquid DAB+substrate chromogen system, Dako Ref K3468). The section were counterstaining with haematoxylin. Negative controls were prepared by replacing the primary antibodies with rabbit IgG. Quantitation of the positive signals in the images was performed using Image J software (Image J, NIH, USA).
Glomerular number was estimated by counting the developed glomeruli in 8–10 different fields for the same kidney section then averaged. One random kidney section was used from 3–4 different biological repeats from each group. Glomerular size was measured using Image J (Image J, NIH, USA) in 8–10 different images for the same kidney section then averaged. Glomerular and tubular structure, in addition to glomerular number and size were additionally assessed by an independent pathologist in a blinded manner for confirmation.