Pk10006

The slices were dewaxed into water, and the endogenous peroxidases were eliminated by adding a 3% hydrogen peroxide solution. The nucleic acid fragments of mRNA were exposed by proteinase K (20 μg/ml) diluted with 3% citric acid. After prehybridization, the hybridization solution containing the VIP mRNA probe (5`-AGTGA GGTTG GATGA CAGGC TGCAG TTCGA AGGAG CAGGT-3`; 5`-ATTAT GATGT GTCCA GAAAT GCCAG GCATG CTGAT GGAGT-3`; 5`-GACAC TCTGA TGCAG TCTTC ACAGA TAACT ACACC CGCCT-3`) was added and and incubated overnight at 37℃. Then, BSA blocking solution was added, followed by mouse anti-digoxigenin-labeled peroxidase (Boster Biological Technology Co., Ltd., China, MK3902-R). After DAB (Proteintech Group, Inc., China, PK10006) and hematoxylin (Beijing Solarbio Science &Technology Co., Ltd., China, G1120) staining, gradient ethanol dehydration, and xylene clearance, they were sealed with neutral glue. Image acquisition and analysis were performed with Image-Pro Plus.

The slices were dewaxed into water, blocked with 5% Albumin from bovine serum (BSA) blocking solution, and 3% hydrogen peroxide solution was added to eliminate the endogenous peroxidase activity. Then the primary antibody (An anti-rat VIP antibody)(Boster Biological Technology Co., Ltd., China, PB1017) was added and incubated overnight at 4℃, and the second antibody (Goat anti-rabbit & mouse Horseradish Peroxidase (HRP) labeled polymer) was added and incubated at room temperature for 30 min. The negative control used phosphate-buffered saline instead of the primary antibody. After Diaminobenzidine (DAB) (Proteintech Group, Inc., China, PK10006) and hematoxylin (Beijing Solarbio Science &Technology Co., Ltd., China, G1120) staining, gradient ethanol dehydration, and xylene clearance, they were sealed with neutral glue. Image acquisition and analysis were performed with Image-Pro Plus.