After being washed three times with PBS, cultured cells were fixed in 4% paraformaldehyde (PFA) for 15 min, and then soaked in a PBS solution containing 0.1 Triton X-100, 1% fetal bovine serum (FBS, GIBCO), and 5% BSA for 1.5 h. Subsequently, the cells were incubated with multiple primary antibodies overnight at 4 °C, and washed three times with PBS on the second day, and then incubated with appropriate secondary antibodies (1:1000, Invitrogen) and DAPI (1:1000, Sigma-Aldrich) in 5% BSA for 1.5 h at room temperature. The primary antibodies include rabbit anti-GFAP (1;500, bs-0199R, Bioss), mouse anti-GDNF (1:500, sc-13147, SANTA CRUZ). The secondary antibodies included donkey antimouse Alexa Fluor488 (A21202, Invitrogen,1:1000), donkey antirabbit Alexa Fluor546 (A10040, Invitrogen, 1:1000). Finally, images were captured with a microscope (Nikon, Tokyo, Japan) at room temperature and analyzed by Photoshop (Adobe) and Image J.
Bs 0199r
Manufactured by Bioss Antibodies
Sourced in China
Bs-0199R is a laboratory equipment designed for various research applications. It is a multipurpose device that can be used for a range of tasks. The core function of Bs-0199R is to assist researchers in their laboratory work, but a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
A21202, by Thermo Fisher Scientific (1 mentions)
A10040, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Imagej, by Adobe (1 mentions)
Cm1850, by Leica (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Goat serum, by Solarbio (1 mentions)
Bs 0127r, by Bioss Antibodies (1 mentions)
Af0693, by Affinity Biosciences (1 mentions)
A10042, by Thermo Fisher Scientific (1 mentions)
Ab45150, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
Bs 0295g, by Bioss Antibodies (1 mentions)
6 protocols using bs 0199r
1
Immunostaining of Cultured Cells
2
GFAP Immunohistochemistry in Mice
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Mice were sacrificed by cardiac perfusion-fixation with PBS and 4% PFA. Brains were fixed in 4% PFA for 24 h and incubated in 30% sucrose at room temperature for 24 h. Brains were embedded in OCT frozen embedding medium (Leica Biosystems, Wetzlar, Germany). Coronal sections were cut at 25 μm on a freezing microtome (CM1850; Leica Biosystems) and stored in PBS at 4°C. Sections were incubated with 5% hydrogen peroxide at room temperature for 15 min and blocked with 5% goat serum (Solarbio, Beijing, China) for 30 min. Afterwards, sections were incubated with the anti-GFAP primary antibody (bs-0199R, 1:200; Bioss, Beijing, China) at 4°C overnight, followed by treatment with the peroxidase-conjugated Affinipure goat anti-rabbit IgG(H+L) secondary antibody (1:200; ZSGB-BIO, Beijing, China) at 37°C for 2 h. The glial fibrillary acidic protein (GFAP) area was observed with a BX51 microscope (Olympus, Tokyo, Japan) and measured using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, USA).
3
Spinal Cord Scar Tissue Analysis via IF Staining
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For IF staining, anesthetized rats were transracially perfused with pre-cooled 0.9% saline and 4% paraformaldehyde in turn. The 6-μm-thick longitudinal frozen sections of the spinal cord samples were manufactured to be probed with anti-glial fibrillary acidic protein (GFAP) antibody (1:500, Bs-0199r, Bioss, Beijing, China) and goat anti-rabbit IgG Alexa 555 (1:500, ab150086, Abcam, Cambridge, United Kingdom) for observing the area of the scar tissue, and the staining results were observed by a Leica fluorescence microscope (DMi8, Leica, Germany).
4
Immunostaining of Brain Tissue Sections
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For brain tissue paraffin sections, after 1 h of dewaxing, antigen retrieval was performed with sodium citrate solution at 90 °C for 15 min, followed by 45 min of blocking with 10% BSA for immunostaining at room temperature, overnight incubation with primary antibodies at 4 °C, and washing three times with PBS, then incubation with appropriate secondary antibodies for 1 h at room temperature. The primary antibodies include rabbit anti-NLRP3 (PAB38738, Bioswamp, 1:200), rabbit anti-MARK4 (AF0693, Affinity, 1:200), rabbit anti-GFAP (bs-0199R, Bioss, 1:200), rabbit anti-Iba1 (A19776, ABconal, 1:200), rabbit anti-BAX (bs-0127R, Bioss, 1:200). The secondary antibody used is donkey anti-rabbit Alexa Fluor568 (A10042, Invitrogen, 1:1000). Images were captured using a fluorescence microscope (Nikon, Tokyo, Japan), and image analysis was performed using Image J.
5
Western Blot Analysis of MYB Protein
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RIPA buffer containing a protease inhibitor cocktail (Beyotime, Jiangsu, China) was used to extract total intracellular protein. Protein samples were then separated via SDS-PAGE, transferred to PVDF membranes (Millipore), and these blots were blocked for 2 h using 5% BSA in TBST. Next, blots were incubated overnight with anti-MYB (1:1000, ab45150, Abcam, MA, USA) or anti-GAPDH (1:1000, bs-0199R, Bioss, Beijing, China) at 4° C. Following incubation with an appropriate secondary antibody, a chemiluminescence detection signal system (Pierce, IL, USA) was employed to detect protein bands.
6
Oligodendrocyte Differentiation Protocol
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OPCs were directionally differentiated into OLs through being incubated in serum-free medium (50 mL DMEM supplement with 1 × N-2 Max, 1 × B-27, 2 mM glutamine, 40 ng/mL triiodothyronine (T3), and 100 U/mL PS) for 3-5 days. DMEM supplement with 1 × N-2 Max, 1 × B-27, 2 mM glutamine, 10 % fetal bovine serum (FBS), and 100 U/mL PS were applied to induce the differentiation of OPCs into type II astrocytes.
OPCs, OLs, and type II astrocytes were identified with immunocytochemical staining. After being fixed with 4 % paraformaldehyde for 30 min, washed with Immunol Staining Wash Buffer for 3 times, and blocked with QuickBlock™ reagent for 60 min, they were incubated with primary antibody overnight at 4 °C, respectively. The primary antibodies were as follows: A2B5 (1:1000, Invitrogen, 433110), O4 (1 μg/Ml, R&D Systems, MAB1326-SP), myelin basic protein (MBP) (1:500, Invitrogen, PA1-46447), and glial fibrillary acidic protein (GFAP) (1:500, Bioss, bs-0199R). After being washed, cells were labeled with secondary antibody including goat anti-mouse IgM (1:1000, Invitrogen, A-21044) or goat anti-rabbit IgG (1:500, Bioss, bs-0295G) for 2 h. Cell nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI) (Solarbio, C0065).
OPCs, OLs, and type II astrocytes were identified with immunocytochemical staining. After being fixed with 4 % paraformaldehyde for 30 min, washed with Immunol Staining Wash Buffer for 3 times, and blocked with QuickBlock™ reagent for 60 min, they were incubated with primary antibody overnight at 4 °C, respectively. The primary antibodies were as follows: A2B5 (1:1000, Invitrogen, 433110), O4 (1 μg/Ml, R&D Systems, MAB1326-SP), myelin basic protein (MBP) (1:500, Invitrogen, PA1-46447), and glial fibrillary acidic protein (GFAP) (1:500, Bioss, bs-0199R). After being washed, cells were labeled with secondary antibody including goat anti-mouse IgM (1:1000, Invitrogen, A-21044) or goat anti-rabbit IgG (1:500, Bioss, bs-0295G) for 2 h. Cell nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI) (Solarbio, C0065).
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