Horseradish peroxidase

The mouse skin sections were deparaffinized, antigen unmasked (or not), incubated in 3% hydrogen peroxide to block endogenous peroxidase, and reacted with primary antibodies (Table 1) overnight at 4 °C, according to the instructions of the manufacturers. The slides were treated with secondary antibodies conjugated with horseradish peroxidase (Nichirei Biosciences, Tokyo, Japan). Images were photographed using a BZ-X710 All-in-One fluorescence microscope (Keyence, Osaka, Japan). Control staining with IgG isotype yielded no positive signal.

The sections and cells were stained with hematoxylin and eosin (HE), alcian blue, alkaline phosphatase, and Oil-red O using a standard protocol. For immunohistochemistry, after incubation overnight with primary antibodies (Supplemental Table 4), a secondary antibody conjugated with horseradish peroxidase (NICHIREI Bioscience, Tokyo, Japan) and a peroxidase substrate, 3,3′-diaminobenzidine tetrahydrochloride (NICHIREI Bioscience), was used for color development for immunohistochemistry. To perform immunocytochemistry of iPSCs, the cells were fixed in 4% paraformaldehyde and were permeabilized with 0.5% saponin in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin. After incubation with primary antibodies (Supplemental Table 3), cyanin3 (Cy3)-conjugated goat anti-mouse immunoglobulin G (IgG; Jackson ImmunoResearch, West Grove, PA, USA) or Cy3-conjugated goat anti-rabbit IgG (Abcam, Cambridge, UK) was used as a secondary antibody. The All-in-One Fluorescence Microscope BZ-X710 (KEYENCE, Osaka, Japan) was used for observation.

Colon tissue samples were fixed in 10% formalin, embedded in paraffin, deparaffinized,
and retrieved as described previously [4 (link)]. The colon
sections were immunostained with various antibodies by using second antibodies conjugated
with horseradish peroxidase (Nichirei Biosciences Inc., Tokyo, Japan). Peroxidase activity
was visualized using 3,3′-diaminobenzidine tetrahydrochloride (Nacalai Tesque, Inc.,
Kyoto, Japan). Counterstaining was performed with hematoxylin and eosin (H & E).
Alcian Blue (pH2.5)/periodic acid-Schiff base (AB-PAS) staining was used for detection of
sugar chains attached to glycoproteins. Histochemical and biochemical analyses were
performed in at least three separate experiments. A group of five WT mice and a group of
five Nox1^-/Y mice were used for each experiment. At least
three colon sections were analyzed, with at least three images examined for each. Twenty
crypts/colon were counted in analyses of histochemical damage, goblet cell damage, and
BrdU/Ki-67 staining. Colon crypt damage was evaluated by the presence of leukocyte
recruitment/infiltration, thickening of the colon wall, and loss or immaturity of goblet
cells, as described previously [21 (link)].