Immunofluorescence was performed as previously described in our laboratory25 (link)26 (link). In brief, the tissue sections were deparaffinized, rehydrated and underwent antigen recovery. Then, the sections were permeabilized with 0.4% Triton X-100 for 10 min and blocked using normal goat serum (Zhongshan Golden Bridge Inc., Beijing, China) for 1 h to eliminate nonspecific staining and incubated in a mixture of rabbit anti-NR4A1 antibody (Proteintech), mouse anti-microtubule-associated protein 2 (MAP2) antibody (Zhongshan Golden Bridge) and chicken anti-astrocyte marker glial fibrillary acidic protein (GFAP, Zhongshan Golden Bridge) antibody or goat anti-Aldehyde Dehydrogenase 1 Family Member L1 (Aldh1L1) antibody (Santa Cruz) overnight at 4 °C. Cells were washed using PBS and incubated with Alexa Fluor-350 goat anti-mouse IgG, Alexa Fluor-488 goat anti-rabbit IgG, and Alexa Fluor-594 goat anti-chicken IgG or Alexa Fluor-594 donkey anti-goat IgG (Zhongshan Golden Bridge) in the dark for 2 h at 37 °C. Cells were washed again in PBS, mounted, sealed, and dried overnight. Finally, the images were captured using confocal laser scanning microscopy (Leica, Wetzlar, Germany).
Normal goat serum (ngs)
Manufactured by Zhongshan Golden Bridge Biotechnology
Sourced in China
Normal goat serum is a sterile, cell-culture tested, and inactivated serum obtained from healthy adult goats. It is commonly used as a general growth supplement and blocking agent in various immunological and cell-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Ix70 inverted microscope, by Olympus (3 mentions)
Confocal laser scanning microscopy, by Leica (3 mentions)
Glial fibrillary acidic protein (gfap), by Zhongshan Golden Bridge Biotechnology (2 mentions)
Fluoview fvx confocal scan head, by Olympus (2 mentions)
Ab31630, by Abcam (2 mentions)
Ab64693, by Abcam (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Fitc conjugated goat anti rabbit igg, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Biotinylated goat anti rabbit igg, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Alexa fluor 546 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
X 650, by Hitachi (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Southern Biotech (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Tcs sp8, by Leica (1 mentions)
Hematoxylin, by Beyotime (1 mentions)
Rat anti mouse cd31 antibody, by BD (1 mentions)
Ab15323, by Abcam (1 mentions)
9 protocols using normal goat serum (ngs)
1
Immunofluorescence Staining of Neural Cells
2
Multicolor Immunofluorescence Imaging of Neuronal and Glial Markers
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Tissue sections (10 μm) were incubated in normal goat serum (Zhongshan Golden Bridge, Beijing, China) for 30 min followed by incubation with a mixture containing a PDE10A antibody (rabbit polyclonal antibody, 1:100), a microtubule-associated protein 2 (MAP2) antibody (chicken polyclonal antibody, 1:100, Zhongshan Golden Bridge) and a glial fibrillary acidic protein (GFAP) antibody (mouse polyclonal antibody, 1:100, Zhongshan Golden Bridge) overnight at 4°C. Sections were washed twice in PBS and incubated with a mixture of DyLight 488-conjugated goat anti-rabbit IgG (1:200, Zhongshan Golden Bridge), DyLight 549-conjugated goat anti-mouse IgG (1:200, Zhongshan Golden Bridge) and DyLight 405-conjugated goat anti-chicken IgG (1:200, Zhongshan Golden Bridge) in a darkroom for 90 min at 37°C. Tissue sections were mounted in 50% glycerol/PBS. Fluorescence was detected using laser scanning confocal microscopy (Leica Microsystems Heidelberg GmbH, Germany) on an Olympus IX 70 inverted microscope (Olympus, Japan) equipped with a Fluoview FVX confocal scan head. To determine the specificity of antibodies, PDE10A antibody (488 channel), GFAP antibody (549 channel) and microtubule-associated protein 2 (MAP2) antibody (405 channel) were replaced by PBS with the same protocols.
3
NEDD9 Immunohistochemistry in Renal Cell Carcinoma
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Immunohistochemistry assay were performed on tissues collected from 68 pathologically confirmed patients with RCC and 6 normal renal tissues. Immunohistochemistry experiments were performed as described previously (11 (link)). In brief, representative samples were placed into 4% paraformaldehyde overnight and then 5-mm paraffin sections were prepared for the experiments. Sections were deparaffinized in xylene, rehydrated which was hydrate by placing in 95, 70, 50 and 30% ethanol for 2 min each, and endogenous peroxidase activity was quenched by 3% hydrogen peroxide in methanol. The sections were submerged in 10 mM citrate buffer (pH 6.0) and microwaved for 8–15 min for antigen retrieval. Non-specific binding was blocked by incubation with normal goat serum (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 1 h at room temperature, and the slides were incubated with NEDD9 rabbit monoclonal primary antibodies (cat. no. ab37161; 1:200; Abcam, Cambridge, MA, USA) at 4°C overnight. After washing, sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG at room temperature for 1 h (cat. no. SPN-9001; 1:50; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.). Color was developed with the DAB Horseradish Peroxidase Color Development kit and imaged using bright field light microscopy.
4
Immunofluorescent Labeling of Rat Brain Cryosections
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The rat cryosections were thawed at room temperature and then fixed in acetone solution for 30 min before staining. After several washes in PBS, the sections were preincubated with 0.4% Triton for 30 min, washed in PBS, and blocked with normal goat serum (Zhongshan Golden Bridge, Beijing, China) at room temperature for 1 h. For double labeling, the tissue sections were incubated with a mixture of anti-Npas4 antibody (1∶100, Novus biological, USA) and anti-MAP2 antibody (1∶100, mouse monoclnal antibody, Boster, Wuhan, China) or mouse anti-GFAP antibody (1∶100, Zhongshan Golden Bridge, Beijing, China) at 4°C overnight. Tissue sections were washed with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1∶50; Zhongshan Golden Bridge, Beijing, China) and Tetramethyl rhodamine isothiocyanate(TRITC)-conjugated goat anti-mouse IgG (1∶50; Zhongshan Golden Bridge, Beijing, China) in the dark room for 90 min at 37°C, then washed with PBS three times for 10 min each, and mounted in 1∶1 glycerol/PBS. Fluorescent images were collected using laser scanning Confocal microscopy (Leica Microsys-tems Heidelberg GmbH, Germany) on an Olympus IX 70 inverted microscope (Olympus, Japan) equipped with a Fluoview FVX confocal scan head.
5
Immunohistochemical Analysis of Fas and FasL
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After 24 hours of reperfusion, rats were anesthetized with 10% (v/v) chloral hydrate and then fixed in 4% (w/v) paraformaldehyde through cardiac perfusion. The right hemisphere was harvested and sliced into paraffin-embedded sections at a thickness of 5 μm. Slices were blocked with normal goat serum (1:10, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), and incubated with rabbit anti-rat Fas and FasL polyclonal antibodies (1:100, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) at 4°C overnight; and then incubated with biotinylated goat anti-rabbit IgG (1:300, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) for 12 minutes at room temperature. Horseradish peroxidase-conjugated streptavidin working solution (1:300) was added and incubated for a further 12 minutes. Negative controls were incubated with PBS instead of antibodies. Three random sections from each rat were examined at five different visual fields under 200× magnification. The MiVnt image analysis system was used to count the number of Fas- and FasL-positive cells in the right hippocampus. The results were averaged.
6
Macrophage Behavior on Scaffolding Biomaterials
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Macrophages were seeded on different membranous scaffolds (PCL, PCL-epECM/H, PCL-epECM/H-IL-4, n = 5) in 48-well plates at a density of 5 × 103 cells/well. After 1 and 3 days of culturing, the macrophages were fixed with 4% PFA (Solarbio, China) and blocked using 5% normal goat serum (Zhongshan Golden bridge Biotechnology, China) for 45 min at room temperature, and then incubated with primary antibodies for mouse anti-CD68 (1:100, Abcam, ab31630, USA), and rabbit anti-Mannose Receptor antibodies (1:200, Abcam, ab64693, USA) overnight at 4 °C. Alexa Fluor 488 goat anti-mouse IgG (1:200, Invitrogen, USA) and Alexa Fluor 546 goat anti-rabbit IgG (1:200, Invitrogen, USA) were applied to react with the primary antibodies for 2 h at room temperature. Finally, the nuclei were stained with DAPI (Southern Biotech, England) and observed with a LSCM (Leica TCS SP8, Germany). Three images were randomly selected in each sample for statistical analysis. To evaluate cell morphology (n = 5), macrophages were fixed by 2.5% glutaraldehyde and dehydrated by gradient alcohol, then were observed under a scanning electron microscope (SEM, Hitachi, X-650, Japan) at an accelerating a voltage of 15 kV. Five cells were randomly selected in each sample for statistical analysis.
7
Immunohistochemical Analysis of CD31 in Tumor Tissues
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Expression of cluster of differentiation 31 (CD31) was measured with a rat anti-mouse CD31 antibody (catalog no., 550674; BD Biosciences). Tumor sections (3–5 µm) of frozen tissues were mounted on 3-aminopropyl triethoxysilane-coated glass slides. Sections were fixed with 4% paraformaldehyde and washed with PBS (pH 7.4). Endogenous peroxide was blocked with 3% H2O2 for 10 min. Following PBS washes, slides were blocked with 5% normal goat serum (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) in PBS for 15 min at room temperature, followed by incubation with primary anti-CD31 (1:400) antibody in blocking solution overnight at 4°C. All slides were subsequently incubated with a 1:200 dilution of biotin-conjugated goat anti-rat secondary antibody (catalog no., PV6004; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) for 15 min at 37°C and streptavidin-biotin complex at 37°C for 15 min. The immunoreaction was visualized using diaminobenzidine peroxide solution (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) and cellular nuclei were counterstained with hematoxylin (Beyotime Institute of Biotechnology). All specimens were evaluated using the Olympus BX600 microscope and Spot Fiex camera. Control samples exposed to secondary antibody alone exhibited no specific staining.
8
Detecting Adenosine A1 Receptor in Epileptic Rat Hippocampus
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To detect the location of adenosine A1 receptor in hippocampus of epileptic rats, immunoreactivity of adenosine A1 receptor was observed by immunofluorescence. Brain hippocampus tissues was fixed in 4 % paraformaldehyde overnight at 4°C, then were respectively put in 20% and 30% graded sucrose solution for 48 h. Followed, 10-μm-thick frozen sections were cut on a freezing microtome and mounted on the polylysine-coated slides for immunofluorescence. The sections were dried at room temperature for 8 min and immersed in acetone for 15 min. After washed with PBS three times (5 min per time), the slices were heated in 0.01 M citric acid (pH 6.0) for 20 min at 92-98°C for antigen recovery. Then, the slices were permeabilized with 0.5 % Triton X-100 and incubated in normal goat serum (Zhongshan Golden Bridge, Inc., Beijing, China) for 30 min. Slices were incubated in rabbit anti adenosine A1 receptor (1:100; Santa) and mouse anti-MAP2 antibody (1:100; Boster, Wuhan, China) overnight at 4°C. After washed with PBS three times (5 min per time), the slices were incubated with anti-rabbit-FITC (green) and anti-mouse-TRITC (red) (1:100, Zhongshan Golden Bridge) for 60 min and mounted with 50 % glycerol and 50 % PBS in the dark. The fluorescence was detected by a laser scanning confocal microscopy (Leica, Heidelberg) on an Olympus IX70 inverted microscope (Olympus, Tokyo, Japan).
9
In Vivo Evaluation of Engineered ECM Scaffolds
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To evaluate the cellularization, vascularisation and immunoregulatory capacity, the control and ECM-C scaffolds were subcutaneously re-implanted into rats for 2 weeks. DAPI staining was used to evaluate the cell infiltration; H&E staining and immunofluorescence staining for von Willebrand factor (vWF) were used to assess vascularisation. To identify macrophage phenotype, immunofluorescence staining was performed with the following antibodies: CD68 (1:100, Abcam, ab31630), a general macrophage marker; CD206 (1:200, Abcam, ab64693), a marker for M2-like macrophages and inducible nitric oxide synthase (iNOS) (1:200, Abcam, ab15323) as a marker for M1-like macrophages. For immunofluorescence staining, frozen sections were incubated with 5% normal goat serum (Zhongshan Golden bridge Biotechnology, China) for 30 min at room temperature. For intracellular antigen staining, 0.1% Triton-PBS was used to permeate the membrane before incubation with serum. Then the sections were incubated with primary antibodies in PBS overnight at 4 °C, followed by incubation with secondary antibody in PBS for 2 h at room temperature.
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