Histofine simple stain max po

Lung tissues collected from euthanized animals were fixed with 4% paraformaldehyde
phosphate buffer solution (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) and embedded
in paraffin wax. Sections (2–4 µm thick) were cut and stained with haematoxylin and eosin
(HE). Immunohistochemical staining was performed by polymer method using Histofine Simple
Stain MAX-PO (Rat) (NICHIREI BIOSCIENCE INC, Tokyo, Japan) or Histofine Simple Stain
MAX-PO (M) (NICHIREI BIOSCIENCE INC). Endogenous peroxidase activity was blocked by 3%
hydrogen peroxide in methanol. Subsequently, sections were incubated with protein block
serum-free reagent (DAKO, Carpinteria, CA, USA) for blocking non-specific reactions. Then
they were incubated with the antisera for 16 hr at 4°C, as primary immunoreaction. The
antisera from rat (1:1,000) were used for sections of guinea pig and mouse, and the
antisera from mouse (1:1,000) were used for sections of rat. Then they were treated with
Histofine Simple Stain MAX-PO (Rat) for tissues of guinea pig and mouse, and with
Histofine Simple Stain MAX-PO (M) for rat tissues for 30 min at room temperature. For
immunohistochemical detection, 3,3′-diaminobenzidine tetrahydrochloride solution (ImmPACT
DAB Peroxidase Substrate; Vector Laboratories, Burlingame, CA, USA) was used.

Colon tissue from each mouse was fixed in 10% formalin in 0.01 M phosphate buffer (pH
7.2) and embedded in paraffin. Sections (3 µm) were stained with
hematoxylin and eosin (H&E) for histopathological examination by light microscopy. We
used a rabbit anti-mouse myeloperoxidase (MPO) polyclonal antibody (Thermo Scientific,
Cheshire, UK) for staining of MPO-positive neutrophils, and a rat anti-mouse macrophage
(F4/80) antibody (Cederlane, Burlington, ON, Canada). MPO staining and F4/80 staining were
performed using Histofine Simple Stain MAX PO (rabbit) and Histofine Simple Stain MAX PO
(rat) (Nichirei, Tokyo, Japan), respectively. For fluorescence staining, we used a goat
anti-mouse IL-13 (R&D Systems, Minneapolis, MN, USA) and Alexa Fluor 546 donkey
anti-goat IgG antibodies (Invitrogen Corporation, Camarillo, CA, USA). For histological
analysis, the numbers of ulcers and infiltrating cell counts were measured using a BIOREVO
BZ-9000 fluorescence microscope (Keyence, Osaka, Japan).

Organs were fixed with 10% formalin in 0.01 M phosphate buffer (pH 7.2) and embedded in
paraffin. Sections (3 µm) were stained with hematoxylin and eosin
(H&E) for histopathological examination by light microscopy. We used a monoclonal
anti-α smooth muscle actin antibody (α-SMA) (Sigma, St Louis, MO, USA), a rat anti-mouse
macrophage (F4/80) antibody (Cedarlane Labs, Burlington, ON, Canada), and a rabbit
anti-collagen III antibody (LSL Co., Ltd., Tokyo, Japan). Staining for α-SMA was performed
using Histofine Simple Stain Max PO (mouse) (Nichirei, Tokyo, Japan). Anti-collagen III
and F4/80 staining were performed using and Histofine Simple Stain Max PO (rat)
(Nichirei).