Cf488a donkey anti mouse igg

Hinokitiol (99%, Figure 1A), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), collagenase type IV, and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Type I collagen was purchased from Corning (NY, USA). The β‐actin antibody, Dulbecco's modified Eagle's medium (DMEM), L‐glutamine‐penicillin‐streptomycin, fetal bovine serum, Pierce™ 1‐Step Transfer Buffer, SuperScript™ IV First‐Strand Synthesis System Kit, and Fast SYBR™ Green Master Mix were purchased from Thermo Fisher (Waltham, MA, USA). The NucleoSpin^® RNA kit was purchased from Macherey‐Nagel (Düren, Germany). Primary antibodies against Bax, Bcl‐2, phospho‐NF‐κB p65 (Ser⁵³⁶), IκBα, cleaved caspase‐3, and PARP were purchased from Cell Signaling (Beverly, MA, USA). The glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody was purchased from GeneTex (Irvine, CA, USA). Hybond‐P polyvinylidene difluoride (PVDF) membrane, the enhanced chemiluminescence (ECL) western blotting detection reagent, horseradish peroxidase (HRP)‐conjugated donkey anti‐rabbit immunoglobulin G (IgG), and the sheep anti‐mouse IgG were purchased from Amersham (Buckinghamshire, UK). CF488A Donkey anti‐mouse IgG and CF594 Donkey anti‐rabbit IgG were purchased from Biotium (Fremont, CA, USA). Hinokitiol was dissolved in 0.1% dimethyl sulfoxide (DMSO) and stored at 4°C.

Morphological and histological analyses of embryos were collected at various times of gestation. For histological analysis, embryos were rinsed with ice‐cold PBS and fixed with 4% paraformaldehyde at room temperature for 10–30 min, dehydrated through graded alcohols, and embedded in paraffin. Embryos were sectioned at 5 μm thickness and stained with haematoxylin and eosin. Sectioned embryos were photographed using a Nikon E800M inverted microscope. For immunofluorescent analysis, paraffin sections were rehydrated, immunostained with cleaved Caspase‐3 antibody (9664, Cell Signalling Technology), Ki‐67 antibody (12202, Cell Signalling Technology). Antigen retrieval was performed according to the individual antibody instructions. CF488A Donkey anti‐Rabbit IgG (20015, Biotium) and CF488A Donkey anti‐Mouse IgG (20,014, Biotium) were used as the secondary antibody. Nuclei were counterstained with 1 μg/ml Propidium Iodide (P4170, Sigma‐Aldrich), washed and mounted.
Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end‐labeling (TUNEL) assay was performed using MEBSTAIN Apoptosis TUNEL Kit Direct (8445, MBL). Briefly, paraffin‐embedded sections were deparaffinized, DNA nick end‐labelled, and counterstained with 1 μg/ml Propidium Iodide and mounted. The slides were analysed using a TCS SP8 confocal microscope (Leica).