Eclpse 80i

Rats were anesthetized with 4% choral hydrate (10 ml/kg) and then perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and post-fixed for 24 hours in the same fixative. Post-fixed brains were embedded in paraffin and sliced on a microtome at a 5 μm thickness. The sections between 2 mm and 3 mm posterior of the bregma were used for this study. For histological assessment of damage to the hippocampus, the paraffin-embedded brain sections were mounted in Poly-L-Lysine-coated slides for histopathological examination by H&E staining. The sections were also incubated in 1% cresyl violet for Nissle staining. To determine CHOP and p-JNK activities, sections were incubated with 0.3% H₂O₂ in methanol for 30 minutes, and then submerged into blocking solution (1% albumin bovine in PBS) for 1 hour at room temperature prior staining. Primary antibodies, such as CHOP (1:200) and p-JNK (1:500) were applied on sections with overnight incubation at 4°C. After 3 times of PBS wash, they were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 hours at 37°C. The reaction was terminated with 3, 3′-diaminobenzidine, and images were captured at 400x magnification using a Nikon ECLPSE 80i. The optical density of CHOP and p-JNK within the injury site in hippocampus were counted at five randomly selected fields per sample [55 (link)].

To determine CHOP, GRP-78, cleaved-PARP and cleaved caspase-12 activities, sections were incubated with 0.3% H₂O₂ in methanol for 30 min., followed by blocking with 1% bovine albumin in PBS for 1 hr at room temperature. Next, the sections were incubated at 4°C overnight with a primary antibody against CHOP (1:200), GRP-78 (1:200), cleaved-PARP (1:200) or cleaved caspase-12 (1:1000). After primary antibody incubation, the sections were washed for 4 × 10 min. at room temperature and then incubated with donkey antimouse/rabbit, donkey antirabbit/mouse or donkey antigoat secondary antibody (1:500; Invitrogen) for 1 hr at room temperature. The saline injection group was considered the negative control. The images were captured using a Nikon ECLPSE 80i.

After deparaffinization and rehydration of skin tissue sections, the endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min at room temperature. After washing with PBS, the nonspecific binding of the samples sites was blocked with 5% BSA for 30 min at 37°C. Subsequently, the sections were incubated at 4°C overnight with antibody against CD31 (1:100), VEGF (1:300), collagen I (1:300), and collagen III (1:300). Afterward, the tissues were washed thrice with PBS and incubated with biotinylated secondary antibodies that diluted with PBS (1:1,000) in 37°C for 1 h. The reaction was stopped by a DAB Chromogen Kit for all sections for 8 s to 3 min, and sections were counterstained with hematoxylin, hydrated, and mounted with a neutral resin. Images were taken using a Nikon light microscope (ECLPSE 80i, Nikon, Japan), and the positive areas of collagen I and collagen III were quantified through Image-Pro Plus 6.0 (Media Cybernetics, Inc., USA).

Tissues were fixed in 4% paraformaldehyde and subsequently embedded in paraffin. Sections (5 μm thick) were stained with hematoxylin and eosin (HE) using a standard protocol and analyzed by optical microscopy (Nikon ECLPSE 80i, Japan) to observed the mesangial cell proliferative. The collagen content of the kidney was detected by Masson’s trichrome kit (Sigma-Aldrich, USA). All above assay were experiment as previous.

To determine the LC3, p-62 and Ub activities, sections were incubated with 0.3% H₂O₂ in methanol for 30 min, followed by blocking with 1% bovine albumin in PBS for 1 h at room temperature. Next, the sections were incubated at 4°C overnight with a primary antibody against LC3 (1:200), p-62 (1:200) or cleaved Ub (1:200). After primary antibody incubation, the sections were washed for 4 × 10 min at room temperature and then incubated with donkey anti-mouse/rabbit, donkey anti-rabbit/mouse, or donkey anti-goat secondary antibody (1:500; Invitrogen) for 1 h at room temperature. The saline injection group was the negative control. The images were captured using a Nikon ECLPSE 80i.

For histopathological examination, gastric tissues were fixed in 4% paraformaldehyde overnight. Selected tissue blocks were processed using a routine overnight cycle in a tissue processor. The tissue blocks were then embedded in wax, serially-sliced into 5 μm sections. The transverse sections were stained with Hematoxylin–Eosin (HE) for tissue damage, visualized and imaged under a light microscope (Nikon ECLPSE 80i, Tokyo, Japan).