Rabbit anti mouse ki 67 clone sp6

Immediately after sacrificing the mice, tissue samples from the proximal, median and distal parts of the colon were collected and fixed overnight in 5% formalin. Paraffin inclusions were performed on the macroscopic injured area. Then, 4 µm sections were stained with hematoxylin-eosin-saffron (HES). A histological evaluation was performed by an experimented pathologist blinded to the nature of the mice being examined, using a five-degree severity score based on inflammatory cell infiltration, erosions and ulcerations, epithelial hyperplasia and mucin depletion, as previously described (29 (link)). Cytofix/Cytoperm buffer (eBioscience) was applied for the detection of nuclear staining. The primary antibodies used for immunostaining of mouse tissue and primary fibroblasts were: rabbit anti-mouse Ki-67 (clone SP6; Abcam), rabbit anti-human α-smooth muscle actin (α-SMA) (polyclonal; Abcam) and Alexa 488-conjugated rabbit anti-vimentin (Clone D21H3; Cell signaling). The secondary antibody was Alexa Fluor 555 goat anti-rabbit IgG (H+L) (Life Technologies). The nuclei were counterstained with DAPI (Sigma-Aldrich). Fluorescence analysis was performed with Olympus IX81 and Fluoview FV1000 software (Olympus). For quantitative analyses, at least four to five representative high-power fields (HPF, 40X) per section were evaluated in a blinded manner.

Mouse liver tissues were fixed in 4% paraformaldehyde and then embedded in OCT (Sakura). 5 μm Frozen sections were prepared using a Cryotome FSE cryostat (Thermo-Fisher Scientific). The tissue sections were incubated in the blocking buffer (5% donkey serum, 0.3% Triton-X 100 in PBS) at room temperature for 1 hr followed by the staining with primary antibodies. The following primary antibodies were used: Rat anti-mouse F4/80 (clone CI:A3-1, ab6640, Abcam), Rabbit anti-mouse Ki67 (clone SP6, ab16667, Abcam), goat anti-mouse Alox15 antibody (clone H-235, sc-32940, Santa Cruz). Then slides were washed and incubated for 1 h with the following secondary antibodies: donkey anti-rat Alexa Fluor 488, donkey anti-goat Alexa Fluor 594 and donkey anti-rabbit Alexa Fluor 647 (Jackson ImmunoResearch Laboratories). Sections were counterstained with 4′6-diamidino-2-phenylindole dihydrochloride (DAPI) before being mounted. All immunofluorescence staining was performed in the dark. Imaging was performed using a Zeiss LSM 880 and images were processed using Zeiss ZEN software.