immunofluorescent staining with anti-GFP and anti-CD63 antibodies

Abstract

This protocol was used for immunofluorescent staining in fixed HeLa cells with anti-GFP and anti-CD63 antibodies, followed by confocal imaging.

plate cells on glass coverslips and grown until they reach 60% conlfuency

fix cells with 4% paraformaldehyde for 20 min at room temperature

permeabilize cells with 0.1% Triton X-100 in PBS for 5 min

block for 1 h with blocking buffer (PBS with 0.5% Tween20, 0.1% BSA, 0.2% FBS)

incubate coverslips with primary antibodies for 2 h at room temperature

(anti-CD63, exbio, 11-343-C100, mouse; anti-GFP, abcam, ab13970, chicken)

wash coverslips 3 times with PBS-T

incubated coverslips 30 min with secondary antibodies

(goat-anti-mouse-AlexaFluor647, goat-anti-chicken-AlexaFluor488)

wash coverslips 3 times with PBS-T

incubate coverslips with DAPI

10.

wash coverslips 3 times with PBS-T

11.

mounte coverslips using

12.

images were acquired using an LSM780 con- focal microscope (Zeiss) with a 10x or 40× objective

13.

colocalization analysis was performed with Fiji plugin Jacop