In brief, cells of each group were fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton-X100, and blocked in phosphate buffer solution (PBS) containing 5% normal goat serum (Beijing Dingguo Changsheng Biotechnology). Cells were incubated with monoclonal antibodies overnight with 4°C. Antibody dilutions were as follows: β III tubulin, 1:200 (Tuj1; Abcam, Cambridge, UK); tyrosine hydroxylase, 1:200 (TH, Abcam, Cambridge, UK); Nestin, 1:200 (Abcam, Cambridge, UK); Neun, 1:200 (Abcam, Cambridge, UK); LXR α receptor, 1:200 (Abcam, Cambridge, UK); LXR β receptor, 1:200 (Gene Tex, Irvine, CA, USA). After extensive washing three times in PBS, suitable secondary antibodies anti-mouse IgG-Alexa Fluor 488, anti-rabbit IgG-Alexa Fluor 488, anti-mouse IgG-Cy3, and anti-rabbit IgG-Alexa Fluor 594 were diluted at 1:200 in PBS, and then suitable secondary antibodies were added and incubated in darkness for 1 h at room temperature. Nuclear stain 4,6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology, Shanghai, China) was then used for nuclear staining.
Normal goat serum (ngs)
Manufactured by Dingguo
Sourced in China
Normal goat serum is a laboratory reagent derived from the blood of healthy goats. It serves as a source of natural proteins, growth factors, and other biomolecules commonly used in various cell culture and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Tyrosine hydroxylase, by Abcam (4 mentions)
Nestin, by Abcam (4 mentions)
Lxr α receptor, by Abcam (4 mentions)
Lxr β receptor, by GeneTex (4 mentions)
Neuronal nuclei (neun), by Abcam (4 mentions)
Confocal laser scanning microscopy, by Leica (1 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
Mouse anti iba1, by Merck Group (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Beyotime (1 mentions)
Rabbit anti tslpr, by Merck Group (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Fish gelatin, by Merck Group (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Bovine serum albumin (bsa), by Biosharp (1 mentions)
Las x software, by Leica (1 mentions)
7 protocols using normal goat serum (ngs)
1
Immunofluorescence Staining of Neural Cells
2
Immunocytochemistry of Neural Markers
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In brief, cells of each group were xed in 4% paraformaldehyde and permeabilized with 0.3% Triton-X100 and blocked in phosphate buffer solution (PBS) containing 5% normal goat serum (Beijing Dingguo Changsheng Biotechnology, China). Cells were incubated with monoclonal antibodies overnight with 4℃. Antibodies' dilutions were as follows: β III tubulin, 1:200 (Tuj1; Abcam, Cambridge, UK); Tyrosine hydroxylase, 1:200 (TH, Abcam, Cambridge, UK); Nestin, 1:200 (Abcam, Cambridge, UK); Neun, 1:200 (Abcam, Cambridge, UK); LXR α receptor, 1:200 (Abcam, Cambridge, UK); LXR β receptor, 1:200 (Gene Tex, USA). After extensive washing for 3 times in PBS, suitable secondary antibodies anti-mouse IgG-Alexa Fluor 488, anti-rabbit IgG-Alexa Fluor 488, anti-mouse IgG-Cy3, anti-rabbit IgG-Alexa Fluor 594 were diluted at 1:200 in PBS, and then suitable secondary antibodies were added and incubated in darkness for 1h at room temperature. Then nuclear stain 4, 6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology, Shanghai, China) was used for nuclear staining.
3
Immunofluorescence Analysis of Neural Markers
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After incubation with normal goat serum (Dingguo, Beijing, China) for 1 h, the sections were then incubated with rabbit anti-TSLPR (1 : 50; Millipore, Burlington, Massachusetts, USA), mouse anti-NeuN (1 : 50; Millipore), mouse anti-GFAP (1 : 50; Millipore), or mouse anti-Iba1 (1 : 100; Millipore) overnight at 4°C. On the following day, the sections were washed with PBS and incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 555-conjugated goat anti-mouse IgG (1 : 50; Beyotime, Beijing, China) in the dark for 1 h at 37°C. The sections were washed again in PBS and the nuclei were stained with diamidine phenylindole. Finally, images were captured using confocal laser scanning microscopy (Leica).
4
Immunocytochemistry of Neural Markers
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In brief, cells of each group were xed in 4% paraformaldehyde and permeabilized with 0.3% Triton-X100 and blocked in phosphate buffer solution (PBS) containing 5% normal goat serum (Beijing Dingguo Changsheng Biotechnology, China). Cells were incubated with monoclonal antibodies overnight with 4℃. Antibodies' dilutions were as follows: β III tubulin, 1:200 (Tuj1; Abcam, Cambridge, UK); Tyrosine hydroxylase, 1:200 (TH, Abcam, Cambridge, UK); Nestin, 1:200 (Abcam, Cambridge, UK); Neun, 1:200 (Abcam, Cambridge, UK); LXR α receptor, 1:200 (Abcam, Cambridge, UK); LXR β receptor, 1:200 (Gene Tex, USA). After extensive washing for 3 times in PBS, suitable secondary antibodies anti-mouse IgG-Alexa Fluor 488, anti-rabbit IgG-Alexa Fluor 488, anti-mouse IgG-Cy3, anti-rabbit IgG-Alexa Fluor 594 were diluted at 1:200 in PBS, and then suitable secondary antibodies were added and incubated in darkness for 1h at room temperature. Then nuclear stain 4, 6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology, Shanghai, China) was used for nuclear staining.
5
Immunofluorescence Staining of ANXA2 in Cells
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The cell culture, group designation, and transfection were the same as above, but the cells were seeded on a sterilized coverglass in each well of six-well plates. The cells were rinsed with phosphatebuffered saline (PBS) three times at 72 h post transfection, fixed in 4% buffered paraformaldehyde for 15 min at room temperature, and treated with 0.3% Triton X-100 for 10 min and 3% H 2 O 2 for 15 min to block the endogenous peroxidase activity. Afterward, the normal goat serum (1:10, Ding Guo Biotech, Beijing, China) was used to block the non-specific binding of immunoglobulin for 30 min. Then the cells were incubated with a rabbit-anti human ANXA2 antibody (1:200) at 37°C for 3 h and a goat antirabbit antibody (1:2,000, Ding Guo Biotech, Beijing, China) at room temperature for 2 h, and then treated with streptavidin/ HRP (5 µg/mL, Ding Guo Biotech, Beijing, China) at room temperature for 30 min. Finally, the cells anchored to coverglasses were displayed by treating them with diamino-benzidine (1 × working solution, Ding Guo Biotech, Beijing, China) for 5-10 min, washed in distilled water briefly, dehydrated in gradient alcohol solutions, cleared with xylene, and observed under a microscope.
6
Immunofluorescence Microscopy Analysis
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Cells were incubated with primary antibodies (FMG-1B #8 and #61, available at dshb.com ), subsequently incubated with a fluorescently labeled secondary antibody and analyzed by confocal microscopy as described previously (Lv et al., 2017 (link)). In brief, cells were plated on 1% poly (ethyleneimine) coated cover glass, decolorized and fixed in methanol at −20°C for 20 min, permeated cells in PBS buffer for 1 hr, and then blocked in 5% BSA (Biosharp), 10% normal goat serum (Dingguo) and 1% fish gelatin (Sigma) in PBS. Incubated the samples with primary antibodies overnight, washed them, and incubated secondary antibody, washed the samples and mounted them on slides with nail polish. The slides were examined with a Leica confocal microscope (SP8). Images were acquired and processed by LAS X software (Leica) and ImageJ software. The 488 nm laser was used YFP excitation wavelength is 510 nm, the emission wavelength is 525 nm and the exposure time is 200 ms.
7
Immunocytochemistry of Neural Markers
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In brief, cells of each group were xed in 4% paraformaldehyde and permeabilized with 0.3% Triton-X100 and blocked in phosphate buffer solution (PBS) containing 5% normal goat serum (Beijing Dingguo Changsheng Biotechnology, China). Cells were incubated with monoclonal antibodies overnight with 4℃. Antibodies' dilutions were as follows: β III tubulin, 1:200 (Tuj1; Abcam, Cambridge, UK); Tyrosine hydroxylase, 1:200 (TH, Abcam, Cambridge, UK); Nestin, 1:200 (Abcam, Cambridge, UK); Neun, 1:200 (Abcam, Cambridge, UK); LXR α receptor, 1:200 (Abcam, Cambridge, UK); LXR β receptor, 1:200 (Gene Tex, USA). After extensive washing for 3 times in PBS, suitable secondary antibodies anti-mouse IgG-Alexa Fluor 488, anti-rabbit IgG-Alexa Fluor 488, anti-mouse IgG-Cy3, anti-rabbit IgG-Alexa Fluor 594 were diluted at 1:200 in PBS, and then suitable secondary antibodies were added and incubated in darkness for 1h at room temperature. Then nuclear stain 4, 6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology, Shanghai, China) was used for nuclear staining.
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