Anti sm22α antibody

Human pulmonary biopsies and lungs paraffin‐embedded sections were deparaffinized and permeabilized (PBS + 0.1% Triton‐X100) before incubation with anti‐Rac‐GTP antibody (NewEast Biosciences, RRID:AB_1961793) (1/1000) overnight at room temperature. After three washes in PBS, sections were incubated for 1 h at room temperature with the secondary Alexa568‐labelled anti‐rabbit antibody (RRID:AB_10563566) (1/1000). Anti‐SM22α antibody (Abcam) (1/500, overnight at room temperature) with a secondary Alexa488‐labelled anti‐mouse antibody (RRID:AB_138404) (1/1000, 1 h at room temperature) were used to localize smooth muscle. To quantify Rac‐GTP levels within SMC, Rac‐GTP fluorescence intensities were measured with Fiji (Fiji, RRID:SCR_002285) inside a mask delimited by SM22α positive cell areas and normalized to the control condition. To emphasize the specificity of Rac‐GTP signal within SMC, an intensity profile was realized on Fiji along the designated line. To allow the comparison between them, each channel intensities was normalized individually as follow: ‐ lowest intensity level = 0 A.U. and highest intensity level = 1 A.U.

Human pulmonary biopsies paraffin-embedded sections were deparaffinised and permeabilised (phosphate buffered saline (PBS)+0.1% Triton-X100) before incubation with anti-Rac–GTP antibody (26903, NewEast Biosciences, King of Prussia, Pennsylvania) (1/1000) overnight at room temperature (RT). After three washes in PBS, sections were incubated for 1 hour at RT with the secondary Alexa568-labelled anti-rabbit antibody (1/1000). Anti-SM22α antibody (Abcam) (1/500 O/N at RT) with Alexa488-labelled anti-mouse antibody (1/1000 1 hour at RT) were used to localise smooth muscle. To quantify Rac–GTP levels within the smooth muscle, Rac–GTP fluorescence intensities were measured inside a mask delimited by SM22a-positive cells and normalised to the control condition.