Alexa fluor 488 conjugated donkey anti rat igg h l

Thymus, embedded with the optimal cutting temperature compound, was cut into 6 μm slides. The slides or cells were fixed for 20 min with 4% polyoxymethylene and blocked in PBS containing 1% BSA. Immunofluorescence staining was performed by the reported standard protocol (31 (link)). For analysis of the thymic medulla and cortex region, the thymic slides were strained via primary antibodies [rabbit anti-KRT5 (Covance; PRB-160P; clone AF 138) and rat anti-KRT8 (DSHB; ab531826; Troma-I)] and followed by the secondary antibodies Alexa Fluor 594-conjugated donkey anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories; 711-586-152) and Alexa Fluor 488-conjugated donkey anti-rat IgG (H+L) (Jackson Immuno Research Laboratories; 712-546-150). For the detection of antinuclear antibodies in 8-month-old WT and Dhx9 cKO mice, Sera were diluted by 1:30 as primary antibodies. The combination was detected via Alexa Fluor 488-conjugated donkey anti-mice IgG (H + L) antibodies (Jackson Immuno Research Laboratories; 715-546-150). Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich; D9542). Images were taken by a laser-scanning N-SIM super-resolution confocal microscope (Nikon, Tokyo, Japan).

Tissues were embedded in optimum cutting temperature compound (Sakura, 4583) and frozen in liquid nitrogen. Sections (6 μm in thickness) and/or cells were fixed for 20 min with 4% polyoxymethylene (Solarbio, P1110) and blocked in PSB containing 1% BSA. Then, sections and/or cells were incubated with primary and secondary antibodies for 1 h at room temperature. Samples were stained with DAPI (1:1,000) after secondary staining. The following antibodies were used for staining: rabbit anti-KRT5 (Covance, PRB-160P; clone AF 138) diluted by 1:400 and rat anti-KRT8 (DSHB, AB 531826; Troma-I) diluted by 1:200. Sera of 8-month-old wild-type and Sirt6 cKO mice (diluted by 1:30) were used as primary antibodies for the detection of the autoantibodies. The secondary antibodies were used for staining: Alexa Fluor 594-conjugated donkey anti-rabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, 711-586-152) diluted by 1:400, Alexa Fluor 488-conjugated donkey anti-rat IgG (H + L) (Jackson ImmunoResearch Laboratories, 712-546-150) diluted by 1:400, and Alexa Fluor 488-conjugated donkey anti-mice IgG (H + L) antibodies (Jackson ImmunoResearch Laboratories, 715-546-150) diluted by 1:300. All antibodies were diluted in 0.5% BSA in PBS. Laser scanning confocal microscope (Zeiss LSM710, Oberkochen, Germany) were used to acquire images.