Fitc labeled goat anti rabbit igg

Immunofluorescence (IF), as an important immunochemical technique, can allow the detection and localization of a wide variety of antigens in different types of tissues. The FFPE blocks collected were sliced (4 μm sections) and deparaffinized in xylene, rehydrated in alcohol, and then washed in distilled water. For antigen retrieval, the sections were microwaved in citrate buffer and blocked using a protein block. The sections were incubated with rabbit anti-S100A8 polyclonal (Beyotime, China) and mouse anti-IL17F monoclonal (Santa Cruz, USA). Subsequently, secondary antibodies FITC-labeled goat anti-rabbit IgG (Beyotime, China) and cy3-labeled goat anti-mouse IgG (Beyotime, China) were added and incubated at room temperature for visualization. The sections were counter-stained with DAPI (Beyotime, China), and then images were captured by an automatic quantitative pathology imaging system (Vectra Polaris, PerkinElmer, Waltham, MA). For performing immunohistochemistry (IHC), the sections were incubated with the mouse anti-S100A8 monoclonal (Immunoway, China) overnight at 4°C, followed by HRP-labeled goat anti-mouse IgG(H+L) (Beyotime, China) treatment at room temperature for 2 hours. Third, 3,3-diaminobenzidine (DAB, Beyotime, China) was used for visualization, and the sections were imaged by a DM1L inverted microscope (Leica, Germany).

Immunofluorescence (IF) assay was used to assess the expression of WT1, LC3II and nephrin protein in kidney tissues. A total of 0.25% Triton X-100 was used to permeabilize 5-μm thick kidney sections for 15 min, and 5% Bovine Serum Albumin (BSA) was used to block them for 1 h at room temperature. Then, the sections were stained with primary antibodies against WT1 (1:200), LC3II (1:200), and nephrin (1:500) overnight at 4°C, followed by secondary Cy3-labeled goat anti-rabbit IgG (A0516, Beyotime), Cy3-labeled goat anti-mouse IgG (A0521, Beyotime) or FITC-labeled goat anti-rabbit IgG (A0561, Beyotime). The cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI), and images were obtained with fluorescence microscopy.

The collected cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, permeabilized with 0.3% Triton X-100 for 30 min, and then blocked by 1% goat serum. After that, cells were treated with anti-TH (Abcam, 1:500) or anti-Iba-1 (Iba-1, Abcam, 1:200) antibody at 4^°C overnight, followed by incubation with secondary antibody FITC-labeled goat anti-rabbit IgG (Beyotime, 1:1,000) at room temperature for 2 h. Fluorescence microscope (CX43, Olympus, Japan) was applied to observe the cell morphology of anti-TH-labeled DA neurons. Numbers of TH-positive neurons were recorded from three randomly selected areas of three parallel wells for each group.

The HH Gli1/2 inhibitor GANT61 and the HH pathway agonist SAG were purchased from MedchemExpress (Monmouth, NJ, USA). The immunofluorescence (IF) staining kits containing Cy3-labeled goat anti-rabbit IgG, FITC-labeled goat anti-rabbit IgG, the 4′,6-diamidino-2-phenylindole (DAPI) reagent, and Fluo-3-AM probe were obtained from Beyotime (Shanghai, China). The anti-KRAS antibody was from Proteintech (Chicago, IL, USA). Anti-ERK1/2, anti-phospho-ERK1/2, anti-JNK, anti-phospho-JNK, anti-p38, anti-phospho-p38, anti-p65, anti-phospho-p65, HRP-conjugated anti-rabbit IgG, and HRP-conjugated anti-mouse IgG antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-CD31 antibody for IF was from Abcam (Cambridge, MA, USA). Anti-β-actin and anti-E-selectin antibodies were obtained from HuaAn Biotechnology Co., Ltd. (Hangzhou, China). The lipofectamine 3000 transfection reagent was obtained from Invitrogen (Carlsbad, CA, USA). Human recombinant TGFβ1 and mouse recombinant TGFβ1 were obtained from the R&D system (Minneapolis, MN, USA). Mouse IL-6 ELISA Kits and Mouse MIP-2 ELISA Kits were purchased from Neobioscience (Shenzhen, China). The has-miR-155 mimics, mimics NC, has-miR-155 inhibitors, inhibitors NC, and the primer kit for has-miR-155 qPCR were purchased from JTSBIO CO.,Ltd. (Wuhan, China).

Zona pellucida-free embryos were washed 3 times in PBS, fixed in freshly prepared 4% paraformaldehyde in PBS, permeabilized in 1% Triton X-100 in PBS, and incubated in blocking solution (1% bovine serum albumin in PBS) for 1 h. For immunolabeling, the embryos were incubated overnight at 4 °C with an anti-H3K9me3 antibody (1:800 dilution; ab8898, Abcam) followed by 3 washes with PBS and a 1-h incubation with a FITC-labeled goat anti-rabbit IgG (1:1000 dilution; Beyotime, Shanghai, China) secondary antibody. The samples were then washed and counterstained with 4,6-diamidino-2-phenylindole (DAPI; Beyotime) to label DNA. Fluorescence was analyzed using a Nikon Eclipse Ti-S microscope equipped with a 198 Nikon DS-Ri1 digital camera (Nikon, Tokyo, Japan).
The cell numbers in blastocysts were estimated by counting nuclei stained using DAPI, and the number of TE cells was determined by counting nuclei positive for CDX2. The cell number of ICM was estimated as the total number of nuclei minus the number of TE nuclei (41 (link), 42 (link)). The blastocyst was incubated with anti-CDX2 (1:200, BioGenex Inc., San Ramon, CA) for detecting the TE, and the secondary antibody was Alexa Fluor Cy3-labeled goat anti-mouse IgG (1:1000 dilution; Beyotime, Shanghai, China).

LX2 cells or primary HSCs were seeded on glass coverslips. After the indicated treatment, cells were fixed with 4% paraformalde-hyde for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and then blocked with 5% bovine serum albumin in PBS for 1 h. After incubation with primary antibodies against NF-κB p65(1:100, NB100-82088, Novus Biologicals, Littleton, USA), α-SMA(1:100, NBP2-33006, Novus Biologicals), or desmin (1:100, NB120-15200, Novus Biologicals) overnight at 4 °C, cells were incubated with Cy3-labeled Goat Anti-Rabbit IgG(1:500, A0516, Beyotime, China) or FITC-labeled Goat Anti-Rabbit IgG (1:500, A0562, Beyotime) for another 1 h. Nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and the images were captured using fluorescence microscopy (FV300, Olympus, Japan).

After a 10–day culture, the morphological change was investigated for differentiation by immunofluorescent staining. The GMSCs were validated by the neuronal genes profiling with NES, TUJ1. The samples were fixed with paraformaldehyde for 30 min and then incubated with primary antibody against NES and TUJ1 (Beyotime, Shanghai, China) at 4 °C, overnight. Then the cells were incubated with appropriate fluorescently labeled Cyc3–labeled goat anti–rabbit IgG and FITC–labeled goat anti–rabbit IgG (Beyotime, Shanghai, China). The mounting solution contained DAPI (Beyotime, Shanghai, China) and was used to mount the cells, which were then observed under a confocal laser microscope (Leica, TCS SP8 SR).