N histofine simple stain mouse max po r

Immunohistochemistry was performed using rabbit monoclonal anti-NeuN antibody (ab177487, Abcam) (dilution 1:500) and N-Histofine Simple Stain Mouse MAX PO (R) (Nichirei Biosciences) with a standard protocol^{73 (link)}.

Kidneys were transversely cut, fixed in 10% neutral buffered formalin, and embedded in paraffin. Deparaffinized sections (3 μm) were prepared by routine procedure. Immunohistochemical staining was performed after antigen retrieval. Anti‐SLC34A1 (NBP2‐13328; Novus Biologicals, Littleton, CO, USA), anti‐CYP27B1 (ABN182; EMD Millipore, Billerica, MA, USA), anti‐CYP24A1 (OABB02030; Aviva Systems Biology, San Diego, CA, USA), and anti‐parathyroid hormone 1 receptor (PTH1R) (HPA007978; Merck KGaA, Darmstadt, Germany) were combined with secondary Ab N‐Histofine simple stain mouse MAX‐PO (R) (414341; Nichirei, Tokyo, Japan). Anti‐megalin (provided by coauthor Dr Saito) was combined with secondary Ab biotinylated anti‐rabbit IgG (Vector Laboratories, Burlingame, CA, USA). Anti‐F4/80 (MCA497GA; AbD Serotec, Raleigh, NC, USA) was combined with secondary Ab N‐Histofine simple stain mouse MAX‐PO (rat) (414311; Nichirei). Counterstaining with hematoxylin was performed on all slides. Finally, samples were imaged using the BZ‐X700 microscope. Information on the Abs used in this study is provided in Table S1. For negative controls, immunohistochemistry was performed without primary Abs (Figure S1); no protein signals were detected, indicating that nonspecific binding of secondary Abs did not occur.

Tissue samples encompassing the gastric ulcer area were immediately fixed in 10% neutral buffered paraformaldehyde, dehydrated with a graded series of ethanol solutions, and embedded in paraffin. The paraffin‐embedded blocks were sliced into 3‐μm sections, and each section was mounted onto a glass slide and stained with haematoxylin and eosin stain (H&E) or processed for immunohistochemistry. For the latter, sections were activated using Histo VT One (Nacalai Tesque) or 0.01 M citrate buffer (pH 6), preincubated with Protein Block Serum‐Free (Dako, Glostrup) and incubated overnight at 4°C with rabbit polyclonal anti‐CD31 antibody (1:200, cat. no. ab28364, Abcam).
Following treatment with primary antibodies, the samples were immersed in 3% hydrogen peroxide (H₂O₂) for 30 min, incubated for 30 min at room temperature with N‐Histofine Simple Stain Mouse MAX PO(R) (Nichirei Bioscience), and immersed in 3, 3′‐diaminobenzine. The sections were counterstained with Mayer’s haematoxylin for 30 s and dehydrated, cleared and mounted.