Mbs462142

IHC analysis was conducted as previously described [27 (link)]. Deparaffinized tissue sections were treated with 3% hydrogen peroxide in methanol for 10 min to remove endogenous peroxidase. Antigen retrieval was performed in sodium citrate buffer (0.1 M) by using a microwave method. The slides were incubated with normal serum to block nonspecific binding and then incubated for 1 h with primary antibodies (1 : 100 to 1 : 200 dilutions) to TGF-β (MBS462142, MyBioSource), CCL-2 (PAB16617, Abnova, Taipei, Taiwan), CXCL1 (PAB8798, Abnova), and CXCL11 (bs-2552R, Bioss). The slides were incubated for 10 min with biotinylated secondary antibodies (PK-7800, Vector Laboratories, Burlingame, CA, USA) and horseradish peroxidase-conjugated streptavidin. The signals were detected by the application of the 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution and counterstaining with Mayer's hematoxylin. To evaluate the staining, after five circles of equal diameter had been drawn on separate areas, without overlapping, the positive cells were counted from four slides per group.

Immunohistochemistry staining on peritoneal sections was conducted using the following antibodies: polyclonal rabbit anti-TGF-b (MBS462142, Mybiosource, USA) at 1:100 dilution, TNF- (ab6671, Abcam, UK) at 1:100 dilution, and VEGF (LS-B7747, LSBio, USA) at 1:100 dilution in the current investigation. The sections were deparaffinized, rehydrated, and treated in a target retrieval solution of Tris–EDTA (pH = 9). To deliver unmasked antigens, samples containing the target retrieval solution were put in a 98 °C heating bath and kept there for 20 min. To halt endogenous peroxidase, samples were treated with hydrogen peroxide (3 percent H2O2) in PBS for 15 min, and normal rabbit serum (5 percent) in PBS was used to avoid nonspecific background staining. The samples were incubated with primary antibodies for one hour. Secondary antibody staining using a goat antirabbit biotinylated antibody (prediluted, Biocare, USA) for 20 min was used to identify primary antibodies. Then, 20-min incubation with prediluted streptavidin horseradish peroxidase (sHRP) (Biocare, USA) was done. Finally, DAB was used as a chromogen to observe antibody binding areas, and Mayer's Hematoxylin (Bio Optica, Italy) was used to counterstain the samples. IHC grading was performed on a scale of 0 to 3 (0: no reaction ǀ 1: mild reaction ≤ 10% ǀ 2: moderate reaction 10–30% ǀ 3: intensive reaction ≥ 30%).