Cy3 labeled goat anti rabbit igg

Sal was procured from Shanghai Tauto Biotechnology (Shanghai, China), the alanine transaminase (ALT), aspartate aminotransferase (AST), and hydroxyproline (Hyp) detection kits were acquired from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). FN, type I collagen (Col I), Bax and Bcl-2 antibodies were obtained from Abcam (Cambridge, United Kingdom). JNK, p-JNK, PARP, and cleaved caspase 3 antibodies were purchased from Cell Signaling Technology (Massachusetts, United States). SphK1, SphK2, S1PR1, S1PR2, S1PR3, CD9, and tumor susceptibility gene 101 (TSG101) antibodies were procured from ImmunoWay Biotechnology Company (Suzhou, China). Antibody against GAPDH was provided by Proteintech (Wuhan, China). TdT-mediated dUTP nick end labeling (TUNEL) Apoptosis Assay Kit, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DPAI), and Cy3-labeled goat anti-rabbit IgG were all bought from Beyotime Institute of Biotechnology (Shanghai, China). The S1P Assay Kit (S1P-ELISA) was delivered by Echelon Biosciences (Salt Lake City, United States).

For identification, the cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 (Beyotime) for 30 min, and rinsed with PBS for 3 times. After treatment with goat serum for 15 min, the cells were incubated with primary antibody against fibroblast-specific protein 1 (FSP1) (1 : 200 dilution; ABclonal, Wuhan, China) or α-smooth muscle actin (α-SMA) (1 : 200 dilution; Abcam) at 4°C overnight. The cells were rinsed with PBS for 3 times and then incubated with Cy3-labeled goat anti-rabbit IgG (1 : 200 dilution; Beyotime) at room temperature for 60 min. At last, the cells were stained by DAPI solution and observed under a microscope.

The cells were plated in 6-well plates, cultured in DMEM including 10% FBS, washed thrice with ice-cold phosphate-buffered saline (PBS, Boster, Wuhan, China), and fixed in 4% paraformaldehyde for 20 min. PBS containing 0.3% Triton X-100 (PBS-T) was added to each well. The cells were blocked with 1% bovine serum albumin (BSA, Sunshine biotechnology, NanJing, China) in PBS-T for one hour at room temperature, and incubated with anti-SLC8A2/NCX2 monoclonal antibody (1:500 dilution, MBL, Japan) at 4 °C overnight. We added Cy3-labeled goat anti-rabbit IgG in an immunofluorescence staining kit (Beyotime, China). Before mounting, the cells were stained with 1’,6-diamino-2-phenylindole (DAPI, Beyotime, China). Then, they were viewed and photographed under a fluorescence microscope (Zeiss Observer A1, Germany).

UCHL3 plasmid (EGFP-UCHL3) (GenePharma, Shanghai, China) were transfected into HCC cells using Lipofectamine 3000 (Thermo Fisher Scientific, L3000-015). Immunofluorescence staining was performed using Vimentin antibody (Proteintech, 10366-1-AP) and Cy3-labeled Goat Anti-rabbit IgG (Beyotime, A0516). Antifade Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, P0131) was used to seal the slices and then photographed under a confocal microscope (Nikon).

Intestinal tissues were freshly isolated and fixed with 4% paraformaldehyde before paraffin embedding. After deparaffination and rehydration, the sections were stained with hematoxylin and eosin (H&E). For immunohistochemistry, tissues were treated with 0.01 M citrate buffer (pH 6.0) in a microwave and then blocked with blocking buffer (Beyotime, Shanghai, China) for 1 h. After incubation with primary antibodies at 4 °C overnight, sections were then immunostained using the SP method. For immunofluorescence staining, after incubation with the primary antibodies, the sections were incubated with Cy3-labeled Goat Anti-Rabbit IgG (1:1000, A0516, Beyotime, Shanghai, China) and counterstained with DAPI. The slides were cover slipped, and the stainings were observed by using a fluorescence microscope (Leica DM4/6B, Leica, Germany). The following primary antibodies were used: anti-VDR (1:200, 12550S, Cell Signaling Technology, Danvers, MA, USA), anti-Ki67 (1:200, ab16667, Abcam, Cambridge, MA, USA), anti-Sox9 (1:100, ab185230, Abcam, Cambridge, MA, USA), anti-Cleaved Caspase-3 (1:2000, 9664S, Cell Signaling Technology, Danvers, MA, USA) and anti-γH2AX (1:400, 9718T, Cell Signaling Technology, Danvers, MA, USA). At least 10 fields of view were captured, and 100 intact crypts were assessed per mouse. The crypt depth and immunopositive cells were quantified using Image J software.

Frozen brain slices and microglia-bearing coverslips mentioned above were immunostained as previously described [80 (link)]. Briefly, after incubation with primary antibodies of p47^phox (1:200, Sigma-Aldrich, St. Louis, MO, USA), PI3Kγ (1:500, Novus, Littleton, CO, USA), Iba1 (1:500, Millipore, Billerica, MA, USA) and NeuN (1:500, Millipore, Billerica, MA, USA), antibody binding was visualized with Alexa Fluor 488-labeled goat anti-mouse IgG and Cy3-labeled goat anti-rabbit IgG (1:200, both from Beyotime Biotechnology, Shanghai, China). Cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Images of the penumbral regions were obtained using a confocal microscope. The Pearson’s correlation coefficient and the numbers of antibody-positive cells were analyzed by a blinded investigator using Image J.