After differentiation, the cells were washed twice in PBS and fixed in PBS with 4% paraformaldehyde (Sigma-Aldrich, Saint Louis, MO, USA; 158127). Then, the cells were washed three times in PBS with 1% BSA (Sigma-Aldrich; A9418), permeabilized with 0.3% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA; T8787)/1% BSA/PBS with 5% normal goat serum (Jackson Immuno Research, Cambridge, UK; 005-000-121) for 45 min, and then incubated with rabbit anti-tyrosine hydroxylase antibody (dilution 1:600; AssayBioTech, Fremont, CA, USA; B0037) or with rabbit anti-choline acetyltransferase antibody (dilution 1:300; Proteintech, Manchester, UK; 20747-1-AP) in 1% BSA/PBS with 5% goat serum overnight at 4 °C. Subsequently, the cells were washed three times in PBS with 1% BSA and incubated with goat anti-rabbit IgG secondary antibody (DY405) (dilution 1:600; LSBio, Seattle, WA, USA; LS-C355899) in 1% BSA/PBS for 1 h in the dark at room temperature. Similar staining was done using a rabbit IgG isotype control (dilution 1:1500; Bioss, Woburn, MA, USA; bs-0295P) to determine any nonspecific binding. The cells were analyzed using flow cytometry (BD FACSAria II; BD FACSDiva Software V6.1.2).
Goat serum
Manufactured by Proteintech
Sourced in China, United Kingdom, United States
Goat serum is a biological product derived from the blood of healthy goats. It is a complex mixture of proteins, hormones, and other biomolecules that can be used as a supplement in cell culture media for in vitro studies.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (3 mentions)
Facsaria 2, by BD (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Normal goat serum (ngs), by Jackson ImmunoResearch (2 mentions)
A9418, by Merck Group (2 mentions)
Rabbit anti choline acetyltransferase antibody, by Proteintech (2 mentions)
Goat anti rabbit igg secondary antibody dy405, by LSBio (2 mentions)
Triton x 100, by Solarbio (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (2 mentions)
Fluorescence microscope, by Nikon (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Goat serum, by Beyotime (2 mentions)
Goat serum, by Abcam (2 mentions)
Fitc conjugated goat anti mouse igg, by Proteintech (2 mentions)
Cryotome fse machine, by Thermo Fisher Scientific (2 mentions)
Mouse anti cytokeratin 8 18 19 monoclonal antibody, by Abcam (2 mentions)
Cy3 conjugated goat anti rabbit igg, by Proteintech (2 mentions)
Rabbit igg isotype control, by Bioss Antibodies (1 mentions)
Normal goat serum (ngs), by Boster Bio (1 mentions)
Dm4000 b led microscope system, by Leica (1 mentions)
14 protocols using goat serum
1
Immunocytochemical Characterization of Cell Cultures
2
Immunofluorescence Staining of PANC-1 and BxPc-3 Cells
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Briefly, 4% paraformaldehyde was added to Stattic-treated PANC-1 and BxPc-3 cells for 30 min for fixation, and the cells were permeabilized with 0.1% Triton X-100 (Solarbio, Dalian, China) for 10 min, and then the cells were blocked with ready-to-use normal goat serum (BOSTER, Wuhan, China) to eliminate nonspecific fluorescence. After washing away goat serum, the cells were hybridized with primary antibodies (Table S1 ) at 4 °C for 24 h, and then with a fluorescent secondary antibody (DyLight 488-green or 594-red, ProteinTech, Wuhan, China). Finally, the cells were stained with DAPI (Solarbio, Dalian, China), and images were captured using a DM4000 B LED microscope system (Leica Microsystems, Wetzlar, Germany).
3
Histopathological and Immunohistochemical Analysis of SADS-CoV in Porcine Intestinal Tissues
4
Immunohistochemical Analysis of Liver Tissue
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Paraffin sections of liver tissue (3 μm) were dewaxed and rehydrated. After being blocked with 3% H2O2 for 10 min, sections were sealed with goat serum (Proteintech) for 20 min. Then sections were incubated with the following primary antibodies overnight at 4 °C: SOAT1 (BOSTER, 1:200), mouse anti-E-cadherin (Proteintech, 1:200), rabbit anti-Vimentin (Bioss, 1:400). The primary antibody in negative group was replaced by PBS. After washing, horseradish peroxidase-polymer anti-mouse/rabbit (Maixin Biotech) was added in all sections for 1 h at room temperature. Lastly, tissues were stained with 3,3’-diamino-benzidine-tetrahydrochloride and counterstained with hematoxylin (Maixin Biotech). All sections were observed with microscope (Olympus). The IHC score was calculated by multiplying the intensity (negative = 0, canary yellow = 1, claybank = 2, brown = 3) and the positive cell percentage scores (<25% = 1, 25–50% = 2, 51–75% = 3, >75% = 4).
5
Immunohistochemical Analysis of SADS-CoV in Porcine Tissues
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Frozen (–80 °C) small intestinal tissues including duodenum, jejunum and ileum taken from the experimentally infected pigs were pre-frozen at –20 °C for 10 min. Tissues were then embedded in optimal cutting temperature (OCT) compound and cut into 8-μm sections using the Cryotome FSE machine (Thermo Fisher Scientific). Mounted microscope slides were fixed with paraformaldehyde and stained with haematoxylin and eosin for histopathological examination.
For immunohistochemistry analysis, a rabbit antibody raised against the SADSr-CoV 3755 N protein was used for specific staining of SADS-CoV antigen. Slides were blocked by incubating with 10% goat serum (Beyotime) at 37 °C for 30 min, followed by overnight incubation at 4 °C with the rabbit anti-3755 N protein serum (1:1,000) and mouse anti-cytokeratin 8+18+19 monoclonal antibody (Abcam), diluted 1:100 in PBST buffer containing 5% goat serum. After washing, slides were then incubated for 50 min at room temperature with Cy3-conjugated goat-anti-rabbit IgG (Proteintech) and FITC-conjugated goat-anti-mouse IgG (Proteintech), diluted 1:100 in PBST buffer containing 5% goat serum. Slides were stained with DAPI (Beyotime) and observed under a fluorescence microscope (Nikon).
For immunohistochemistry analysis, a rabbit antibody raised against the SADSr-CoV 3755 N protein was used for specific staining of SADS-CoV antigen. Slides were blocked by incubating with 10% goat serum (Beyotime) at 37 °C for 30 min, followed by overnight incubation at 4 °C with the rabbit anti-3755 N protein serum (1:1,000) and mouse anti-cytokeratin 8+18+19 monoclonal antibody (Abcam), diluted 1:100 in PBST buffer containing 5% goat serum. After washing, slides were then incubated for 50 min at room temperature with Cy3-conjugated goat-anti-rabbit IgG (Proteintech) and FITC-conjugated goat-anti-mouse IgG (Proteintech), diluted 1:100 in PBST buffer containing 5% goat serum. Slides were stained with DAPI (Beyotime) and observed under a fluorescence microscope (Nikon).
6
Immunofluorescence Analysis of CD133 and Jagged1
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Cells were cultured on coverslips, washed in cold PBS, fixed in 95% ethanol for 30 min, and permeabilized in 0.3% Triton X-100 (Solarbio, Beijing, China) for 15 min. Fixed cells were blocked in PBS containing 5% goat serum (ZSGB, Beijing, China) at 37 °C for 1 h. Cells were incubated with the primary antibody CD133 (1:1000, #66666-1-lg, Proteintech, Wuhan, China) and Jagged1 (1:400, #bs-1448R, Bioss, Beijing, China) at 4 °C overnight. The secondary antibody goat anti-rabbit IgG (Cy3, #GB303, Servicebio, Wuhan, China) was added on cells which were washed three times at 37 °C in the dark for 1 h. DAPI (Solarbio, Beijing, China) was added on cells for 5 min. The fluorescent signal was captured using a TS100 inverted fluorescence microscope (Nikon, Japan).
7
Quantifying GABAergic Interneuron Differentiation
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Neurons were infected with AAV-scramble-GFP or AAV-shBrpf1-GFP virus at DIV3. The shBrpf1 sequence was shown in Table 1 . After DIV10, GFP-expressing neurons could be gradually observed under a fluorescent microscope. At DIV14-15, immunofluorescent staining was performed to quantify the number of GABAergic interneurons that differentiated into PV+ or SST+ interneurons. Briefly, neurons were fixed in 4% paraformaldehyde (Biosharp, BL539A) in PBS (Hyclone, SH30243.01) for 20 min, permeabilized with 0.1% Triton X-100 (Sigma, X-100) in PBS for 1 h, and blocked with 10% goat serum (Proteintech, b900780) in PBS for 1 h at room temperature. Neurons were then incubated with primary antibodies at 4° overnight and second antibodies for 1 h. The primary antibodies used were mouse anti-MAP2 (1:1000, RRID: AB_2882331), rabbit anti-GFP (1:500, RRID: AB_11042881), mouse anti-GABA (1:200, RRID: AB_476667), rabbit anti-PV (1:500, RRID: AB_2880541), rabbit anti-SST (1:500, RRID: AB_2195910), and mouse anti-GFP (1:500, RRID: AB_11182611). Neurons were finally incubated with DAPI (Sigma, D9542) at room temperature for 10 min and mounted with antifade mounting medium.
8
Immunocytochemistry for Dopamine and Acetylcholine Neurons
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Indirect labeling with a specific anti-TH or anti-CHAT antibody was used to identify the TH or CHAT protein-positive cells. After the differentiation, the cells were washed twice in PBS and fixed in PBS with 4% paraformaldehyde (Sigma-Aldrich; 158127). The cells were then washed three times in PBS with 1% BSA (Sigma-Aldrich; A9418), permeabilized with 0.3% Triton X-100 (Sigma-Aldrich; T8787)/1% BSA/PBS with 5% normal goat serum (Jackson Immuno Research, Cambridge, UK; 005-000-121) for 45 min and incubated with rabbit anti-tyrosine hydroxylase antibody (dilution 1:600; AssayBioTech, Fremont, CA, USA; B0037) or with rabbit anti-choline acetyltransferase antibody (dilution 1:300; Proteintech, Manchester, UK; 20747-1-AP) in 1% BSA/PBS with 5% goat serum overnight at 4 °C. The cells were washed three times in PBS with 1% BSA and incubated with goat anti-rabbit IgG secondary antibody (DY405) (dilution 1:600; LSBio, Seattle, WA, USA; LS-C355899) in 1% BSA/PBS for 1 h in the dark at room temperature. In order to determine any nonspecific binding, a similar staining was conducted using a rabbit IgG isotype control (dilution 1:1500; Bioss Antibodies; bs-0295P). Flow cytometry was used to analyze the cells (BD FACSAria II; BD FACSDiva Software V6.1.2).
9
Immunofluorescence Staining of Cells and Cardiac Tissue
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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).
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).
10
Immunohistochemical Analysis of IL-9 and IL-9R
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All skin samples were 10% formalin-fixed, paraffin-embedded, and cut at 5 µm. After dewaxing and hydration, antigen retrieval was performed in Tris-ethylenediaminetetraacetic acid buffer (pH 9.0) in a pressure cooker. The slices were treated with a 3% H2O2 solution to block endogenous peroxidase activity and, subsequently, incubated with 5% goat serum (Proteintech, Wuhan, Hubei, China) for 1 h at 22°C. Subsequently, mouse IL-9 monoclonal antibody (1:400 dilution; Cat. no. 66144-1-Ig; Proteintech) and rabbit IL-9R polyclonal antibody (1: 200 dilution; Cat. no. PA5-86324; Invitrogen, Carlsbad, CA, USA) were applied overnight at 4°C. After incubation with an HRP-conjugated secondary antibody (Proteintech) at 22°C for 1 h, the tissue slides were stained with diaminobenzidine and photographed (Digital Slide Scanning System PRECICE 500B; UNIC Technologies, Inc., Suzhou, Jiangsu, China).
The results were quantitatively analyzed according to the extent and intensity of immunostaining by assessing the mean value of integrated optical density (IOD) with the support of image-pro plus software (version 16.0; Media Cybernetics, Inc., Rockville, MD, USA). Five areas of each tissue slide (400×) were randomly selected for IOD calculation, and calibration was done before calculation.
The results were quantitatively analyzed according to the extent and intensity of immunostaining by assessing the mean value of integrated optical density (IOD) with the support of image-pro plus software (version 16.0; Media Cybernetics, Inc., Rockville, MD, USA). Five areas of each tissue slide (400×) were randomly selected for IOD calculation, and calibration was done before calculation.
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