Alexa fluor 594 affinipure goat anti mouse igg

Protein retention by the ER was visualized by fluorescence microscopy using an antibody against the KDEL peptide, present in ER targeted proteins, as described previously.41, 42 Calvaria‐derived osteoblasts were cultured on collagen‐coated cover slips in 6‐well plates and fixed with 4% paraformaldehyde in PBS for 30 minutes at 4°C. Following permeabilization with solution containing 0.2% Triton X‐100, 100 µg/mL BSA, 0.01% sodium azide the cells were stained with anti‐KDEL antibody (1:200, ab12223, Abcam) in PBS containing 100 µg/mL BSA at room temperature for 1 hour. After 3 washes with 0.2% Triton X‐100, cells were incubated with Alexa Fluor®594 AffiniPure Goat Anti‐Mouse IgG (1:100, #115‐585‐003, Jackson Immunoresearch) for 1 hour and stained with DAPI. Images were captured as Z‐stacks with Zeiss LSM 880 Confocal Microscope using a 20X objective with constant parameters of acquisition (excitation wavelength: 405 and 561 nm). The z‐stacks were processed into a single 2D image using the Zen software. KDEL immunostaining was quantified using Image J software. First, a region of interest was selected by manually drawing the cell margin for each cell. Then, the average fluorescence pixel intensity of each cell in the red channel was determined.

The primary antibodies used for Immunofluorescence staining in this study were anti-CD31 (1:300, Abcam, ab222783), anti-ZO-1 (1:300, Abcam, ab221547), anti-Occludin (1:300, Abcam, ab216327), anti-NeuN (1:300, Abcam, ab104224), anti-GFAP (1:1000, Abcam, ab7260), anti-Sox17 (1:100, Santa Cruz Biotechnology, sc-130295), and anti-CD68 (1:300, Abcam, ab283654). The secondary antibodies used for immunofluorescence staining in this study were Alexa Fluor® 488 AffiniPure™Goat Anti-Rabbit IgG (H + L)(1:300, Jackson, 111-545-003) and Alexa Fluor® 594 AffiniPure™Goat Anti-Mouse IgG (H + L)(1:300, Jackson, 115-585-003). Primary antibodies used for western blot studies were anti-ZO-1 (1:1000, Abcam, ab221547), anti-Occludin (1:1000, Abcam, ab216327), anti-β-actin (1:2000, Servicebio, Wuhan, China), anti-Sox17 (1:500, Santa Cruz Biotechnology, sc-130295; 1:1000, Proteintech, Wuhan, China, 24903-1-AP), anti-CD31(1:1000, Abcam, ab222783), anti-UCHL1 (1:1000, Abcam, ab108986), anti-Flag-Tag (1:2000, Cell Signaling Technology, #14793), anti-Myc-Tag (1:2000, Cell Signaling Technology, #9402), and anti-HA-Tag (1:2000, Cell Signaling Technology, #3724). The secondary antibodies used for western blot studies were HRP conjugated Goat Anti-Rabbit IgG (H + L) (1:5000, Servicebio, GB23303) and HRP conjugated Goat Anti-Mouse IgG (H + L) (1:5000, Servicebio, GB23301).

Dewaxed and rehydrated slides of colon sections were boiled in citrate-EDTA antigen retrieval solution for 10 min, and were blocked with 10% normal goat serum. And then incubated with rabbit occludin primary antibody (Proteintech, 27260-1-AP, 1:200) or mouse BRD4 primary antibody (Proteintech, 67374-1-Ig, 1:100) overnight at 4°C followed by Alexa Fluor 488-Affinipure Goat Anti-Rabbit IgG (Jackson, JAC-111-545-144, 1:1,000) or Alexa Fluor 594-Affinipure Goat Anti-Mouse IgG (Jackson, JAC-111-545-144, 1:1,000). 4’,6-dia-midino-2-phenylindole (DAPI) was used for DNA counterstain.