Alexa fluor 568 labeled donkey anti rabbit igg

The samples were fixed in 4% paraformaldehyde, rinsed with phosphate-buffered saline (Beyotime Biotechnology, Nantong, China), blocked with 0.3% Triton-X 100 and 3% bovine serum albumin, and incubated at 4°C overnight with primary antibodies—mouse anti-glial fibrillary acidic protein (1:400; Abcam, Cambridge, UK, Cat# ab21837) and rabbit anti-fibronectin (1:200; Abcam, Cat# ab23751). After rinsing with phosphate-buffered saline, the samples were incubated with secondary antibodies—Alexa Fluor 488-labeled donkey anti-mouse IgG (1:1,000; Thermo Fisher, Cat# A21202) or Alexa Fluor 568-labeled donkey anti-rabbit IgG (1:1,000; Thermo Fisher, Cat# A10042) at room temperature for 2 hours in the dark. Finally, the specimens were sealed with 4′,6-diamidino-2-phenylindole-containing sealant (Vector Laboratories, Burlingame, CA, USA) and observed under a laser-scanning confocal microscope (SP7, Leica, Wetzlar, Germany).

The ability of mAbs 23A1 and 31A5 to detect mLPL was tested on transfected cells and sections of mouse heart. CHO cells were transfected with an expression vector for mLPL and then plated on glass coverslips. The next day, the cell-coated coverslips were fixed in methanol. Frozen heart sections (10-μm–thick) were fixed in 3% paraformaldehyde. After permeabilization in 0.2% Triton X100, samples were blocked in PBS containing 0.2% BSA and 5% donkey serum and then incubated overnight with mAbs 23A1 or 31A5 and the rabbit pAb 3174 against mouse LPL (all at 15 μg/ml). After washing, samples were incubated for 30 min at room temperature with Alexa Fluor 568–labeled donkey anti rabbit IgG (1:200; Thermo Fisher scientific) and DyLight 650–labeled donkey anti rat IgG (1:200; Thermo Fisher scientific). After washing, samples were post-fixed in 3% paraformaldehyde. Cell coverslips were mounted on slides with ProLong Diamond Antifade mountant with DAPI (Thermo Fisher scientific). Images were acquired on a LSM980 Zeiss confocal microscope with a 20× objective lens.