Alexa fluor 488 affinipure donkey anti rabbit igg

Distal ileal sections were heated at 60°C for 1 h. Next, sections were put in Leica Autostainer XL (Leica, USA) (xylene for 40 min, 100% ethanol for 10 min, 95% ethanol for 10 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and doubly distilled water for 3 min) to deparaffinize and rehydrate the samples. Antigens were retrieved using a citrate antigen retrieval solution (Sangon Biotech, China). After repeated washing in phosphate-buffered saline (PBS), a super pap pen (Sangon Biotech, China) was used to draw a circle around the tissue. Slides were blocked with immunostaining blocking buffer (Sangon Biotech, China) at room temperature for 1 h and incubated with primary antibodies against PCNA (catalog number A0264; Abclonal, China), occludin (catalog number A2601; Abclonal, China), claudin1 (catalog number ab211737; Abcam, USA), and ZO-1 (catalog number 13663; Cell Signaling Technology, USA) diluted with primary antibody dilution buffer (Sangon Biotech, China) at 4°C overnight. Slides were washed with PBS and incubated with Alexa Fluor 488 AffiniPure donkey anti-rabbit IgG (Yeason, China) for 1 h at room temperature. Next, the slides were washed with PBS and stained with dihydrochloride (Yeason, China) for 10 min. Images were captured with a fluorescence microscope (Leica, USA).

HTR8/SVneo were plated into six-well plates and fixed with 4% paraformaldehyde for 20 min, and then permeabilized with 0.5% Triton X-100 at room temperature for 20 min. For immunofluorescence, the cells were blocked with 5% BSA for 30 min and then incubated with primary antibody against N-cadherin, E-cadherin (Cell Signaling Technology, Inc, Boston, USA), vimentin, snail, β-TrCP (Abcam, Cambridge, USA)at 4 °C overnight. This was followed by the application of fluorescent secondary antibody (Alexa Fluor 488 Affinipure donkey anti-rabbit IgG or Alexa Fluor 596 Affinipure donkey anti-rabbit IgG) (Yeasen biotech, Co., Ltd, Hong Kong) for 1 h in darkness. The nuclei were stained with DAPI (1:100; Sigma-Aldrich) at 37 °C. Positive staining was observed under a fluorescence microscope.