Histostain plus ihc staining kit

Mice were deeply anesthetized with urethane (1.5 g/kg) and perfused transcardially with PBS followed by 4% paraformaldehyde (PFA) as described [59 (link)]. Brains, left lateral lobes of liver, and left lungs were removed and post-fixed in 4% PFA before dehydration and embedding in paraffin. Sagittal sections from one individual mouse brain were stained with an antibody against the PC marker calbindin-D-28K (Sigma-aldrich, St. Louis, MO) using a Histostain-Plus IHC staining kit (Invitrogen; Grand Island, NY). Cells stained positive for calbindin D in the cerebellum were enumerated from 3 random 10× fields of view under an Imager M2 microscope (Zeiss; Thornwood, NY) in a blind manner, and three serial sections were scored and the average score of each mouse cerebellum was calculated. Lung and liver tissue sections were stained with anti-Ki67 antibody (Biocare Medical; Concord, CA) using a Histostain-Plus IHC staining kit (Invitrogen; Grand Island, NY). Ki67-positive cells were scored under an Imager M2 microscope (Zeiss; Thornwood, NY) in a blind manner. In lungs, Ki67-positive cells in the bronchial epithelium and alveoli were scored from 4 random 10× and 4 random 20× microscopic fields of view, respectively, and the percentage of Ki67-positive cells was calculated. In liver, Ki67 positive cells were enumerated from 4 random 10× fields of view.

Liver tissue sections were prepared as described above and stained with a cleaved-caspase 3 antibody (Cell Signaling; Beverly, MA, USA; catalog number 9664) or 4-hydroxynonenal (4-HNE) antibody (Abcam; Cambridge, MA, USA; catalog number ab46545) using a Histostain-Plus IHC staining kit (Invitrogen; Grand Island, NY, USA). After counter staining with hematoxylin, cells positive for the cleaved caspase 3 or 4-HNE staining were numerated in five 20× random fields in each tissue section under the Imager M2 microscope in a blind manner. TUNEL assays were performed using TACS® 2 TdT diaminobenzidine kit (Trevigen; Gaithersburg, MD, USA) according to the manufacturer’s instructions, methyl green was used for counter staining. TUNEL-positive cells were numerated as described above.