Alexa fluor 488 conjugated affinipure goat anti rabbit igg

The THP-1 macrophages were fixed with 4% paraformaldehyde (Sigma) followed by permeabilization with 0.2% Triton X-100 (Thermo Fisher Scientific). Cells were blocked with 3% BSA and labelled with Rabbit polyclonal to LC3 antibody (Abcam, ab51520) and visualized by Alexa Fluor 488-conjugated Affinipure Goat Anti-Rabbit IgG (Proteintech). Nuclei were stained with DAPI. The fluorescence images of cells were acquired and examined using a confocal microscope (Olympus, Japan). To quantify autophagy, the number of LC3 punctate dots was calculated by ImageJ Software (Version 1.49). At least 10 cells per experimental group were counted and each condition was assayed in triplicate.

To determine the functional pathway of PSMB9, the intracellular localization of PSMB9 was detected by immunofluorescence assay. SW1088 and SW1783 cells were planted on slides, immobilized with 4% paraformaldehyde for 60 min until the cells grew into a suitable density, washed with PBS several times to remove the residual paraformaldehyde, and then incubated in 0.3% Triton X-100 (dissolve in PBS) for 15 min. The cells were washed with PBS another three times, and the antigen was blocked with 5% goat serum for 1 h. Then a rabbit anti-PSMB9 antibody (14544-1-AP; 1:50; proteintech, Wuhan, China) was used to especially bind the PSMB9 protein in the cells at 4°C for no less than 12 h. Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG was used as the secondary antibody to conjugate with the primary antibody after the primary antibody incubation is complete. Next, the sample needs to be incubated with 10 μg/ml of DAPI for 20 s in the dark. Finally, the sample was washed three times with PBS, and anti-quench sealing tablets were used for anti-quench treatment (Fluorescent Mounting Media; Bioss, Woburn, M, USA; Cat. C02-04003) if necessary, followed by confocal microscopy (Leica, Wetzlar, Germany).

Cells were seeded on a 24-well plate and cultivated until the confluence reached 70%~80%. Cells were washed with PBS three times, fixed with 4% paraformaldehyde (Sangon, Shanghai, China) for 15 min at room temperature, with subsequent permeation with 0.5% Triton® X-100 (Sigma, Shanghai, China) for 20 min. Normal goat serum (Beyotime, Shanghai, China) was added to the cells and closed at room temperature for an hour. The cells were incubated with primary antibody against ALKBH5 (Proteintech, Wuhan, China) and cTNT (Proteintech, Wuhan, China) overnight for 4°C and Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit IgG (Proteintech) for 1 h at 37°C. The cell nucleus was stained with DAPI (Sigma) away from the light for 5 min. Photographs were acquired utilizing a fluorescence microscope (Zeiss, Baden-Wurtberg, Germany).
The procedure for immunofluorescence (IF) staining of cardiac tissue sections paralleled that of IHC, with the distinction that after antigen retrieval, sections were blocked using 5% goat serum, followed by overnight incubation with primary antibodies (Proteintech, Wuhan, China). Subsequently, the sections were treated with Alexa Fluor-tagged secondary antibodies for 1 h at ambient temperature and then mounted in a medium infused with DAPI (Sigma).

One drop (30–50 μl) of the diluted mouse antibody to SFTSV recombinant NP as previously mentioned was added. Negative controls were added with normal mouse sera without the primary antibody correspondently. The sections were incubated at 37 °C for 1 h. The slides were rinsed with PBS. For the immunohistochemistry assays, the sections were incubated with rabbit anti-mouse horseradish peroxidase-labeled secondary antibody (1: 1000) (ZSGB-Bio, Beijing, China), and incubated at 37 °C for 1 h. The sections were stained with DAB coloring liquid (ZSGB-Bio, Beijing, China) and observed under a light microscope by two pathologists. For the indirect immunofluorescence assays, the sections were incubated with rabbit anti-mouse peroxidase-labeled secondary antibody (Alexa Fluor 488-conjugated Affinipure Goat Anti-Rabbit IgG, Proteintech, USA) (1:100 diluted), and then placed in an incubator at 37 °C for 1 h. After immunoreaction, nucleus staining with DAPI (C0065, Solarbio) was performed for 10 min at room temperature. The slides were washed again and the slides were examined under a fluorescence microscope (Olympus BX3).