Alexa fluor 488 conjugated affinipure donkey anti goat lgg h l

The cardiac tissues in different groups were prepared into frozen sections for immunofluorescence analysis. The 4.5-um frozen section and cardiac fibroblasts were fixed for 20 min in 4% paraformaldehyde (PFA) at room temperature. After fixation, they were blocked with 5% bovine serum albumin (BSA) in Tris Buffered Saline With Tween (TBST) for 1 h, and then incubated with appropriate primary antibodies at 4 °C over night and incubated with second antibodies (Alexa Fluor ®488-conjugated affinipure goat anti-rabbit lgG (H+L), Cy^TM 3-conjugated affinipure donkey anti-goat⁺⁺ lgG (H+L), and Alexa Fluor ®488-conjugated affinipure donkey anti-goat++ lgG (H+L) (Jackson ImmunoResearch Laboratories, Inc., PA, USA)) for 1 hour at room temperature. After that, sections and cells were stained with 1.5 μM 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Sigma, St. Louis, Missouri, USA) for 10 min. The image analysis of frozen sections was performed using a software program (Olympus, Japan). Protein localization of cells was observed and captured with a laser scanning confocal microscope (LSM5, Zeiss, Jena, Thuringia, Germany).

Primary antibodies: TMEM16A antibody (Santa Cruz Biotechnology, Inc., Texas, USA), α-smooth muscle actin (α-SMA) antibody and TGF-β1 antibody (Epitomics, Inc., California, USA), Smad3 antibody and Smad3 (phospho S423+S425) antibody (Abcam, Inc., Cambridge,UK), Vimentin antibody(Abcam, Inc., Cambridge,UK), and Collagen I antibody (wanleibio co.,ltd, Shenyang, China), and Gapdh antibody (HuaAnBiotech, Inc., Hangzhou, China).
Secondary antibodies: horseradish peroxidase-conjugated rabbit-anti-goat IgG antibody (HuaAnBiotech, Inc., Hangzhou, China), peroxidase-conjugated affinipure goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc., PA, USA), Alexa Fluor ®488-conjugated affinipure goat anti-rabbit lgG (H+L), Cy^TM 3-conjugated affinipure goat anti- mouse⁺⁺ lgG (H+L), and Alexa Fluor ®488-conjugated affinipure donkey anti-goat++ lgG (H+L) (Jackson ImmunoResearch Laboratories, Inc., PA, USA).