实验是很多临床同学永远的痛，相比基础的同学，也许是先天不足，而对于基础实验中最基本的WB应该是必须知道的，今天就向大家介绍-----------western blot。western blot。二抗带 HRP或 AP标记。
1)、石蜡切片脱蜡至水:依次将切片放入环保型脱蜡液Ⅰ 10min——环保型脱蜡液Ⅱ 10min——环保型脱蜡液Ⅲ 10min——无水乙醇Ⅰ 5min——无水乙醇Ⅱ 5min——无水乙醇Ⅲ 5min——蒸馏水洗。
IL-6， IL-1β and TNF-α expression levels were measured to evaluate the inflammatory response in spinal cord tissue and cells. NLRP3， apoptosis-associated speck-like protein containing a CARD ， pro-Caspase-1， Caspase-1， GSDMD， and IL-18 levels were measured to evaluate pyroptosis. Nrf2 and HO-1 levels were measured to assess the Nrf2/ HO-1 signaling axis. First， 100 mg of spinal cord tissue was washed with PBS， fully lysed with radioimmunoprecipitation assay buffer containing phenylmethylsulfonyl fluoride for 30 minutes， incubated on ice， and immediately centrifuged at 4°C. The supernatants were then collected， and the protein concentrations was determined using the bicinchoninic acid method with the PierceTM BCA Protein Assay Kit . The samples were loaded onto a polyacrylamide gel and electrophoresed. Next， the proteins were transferred to polyvinylidene fluoride membranes and blocked in 5% non-fat milk for 2 hours. Subsequently， the membranes were incubated overnight at 4°C with rat monoclonal primary antibodies against IL-6 ， IL-1β ， T N F - α ， NLRP3 ， ASC ， pro-Caspase-1 ， C a s p a s e - 1 ， GSDMD ， and IL-18 ， followed by incubation with horseradish peroxidase–conjugated rabbit anti-rat IgG at 20°C for 1 hour， and finally stained with a diaminobenzidine solution. A Bio-Image gel imaging system was used to collect images. The optical density was measured using ImageJ software. Glyceraldehyde-3-phosphate dehydrogenase and β-actin were used as internal references.背景和对比度不应该加以修改以明显改变数据可见度、背景或非特异性带。
