An immunofluorescence assay was performed as described previously [25 (link)]. Briefly, slices soaked in sodium citrate buffer were heated in a microwave for 6 min three times for antigen retrieval. Then, the slices were cooled to room temperature, washed with 1× PBS containing 0.05% Tween-20 (1× PBST), and incubated with 0.1% Triton X-100 at room temperature for 10 min. The sections were then washed, treated with 3% hydrogen peroxide at room temperature for 10 min, washed again, and sealed with 10% bovine serum albumin at 37 °C for 60 min. The primary antibody was diluted in bovine serum, and the samples were cultured with the antibody at 4 °C overnight or at 37 °C for 60 min. Then, the slices were washed and incubated with the secondary antibody in BSA for 60 min at room temperature. The primary antibodies used were a rabbit monoclonal anti-CD4 antibody (ab133616, Abcam, Cambridgeshire, United Kingdom, 1:1000) and a rabbit polyclonal anti-CD8 antibody (ab4055, Abcam, Cambridgeshire, United Kingdom, 1:400), and the secondary antibodies were goat anti-rabbit (Alexa Fluor 555, ab150078, Abcam, Cambridgeshire, United Kingdom, 1:2000) and goat anti-rabbit (Alexa Fluor 488, ab150077, Abcam, Cambridgeshire, United Kingdom, 1:3000) antibodies. Finally, a Leica DMI4000B Microsystems microscope (Leica Microsystems, Wetzlar, Germany) was used for observation.
Ab150078
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab150078 is a primary antibody that recognizes the GAPDH protein. This antibody is developed for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab150077, by Abcam (12 mentions)
Ab150113, by Abcam (8 mentions)
Lsm 700, by Zeiss (6 mentions)
C1006, by Beyotime (5 mentions)
Ab150114, by Abcam (3 mentions)
Fluorescence microscope, by Leica (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Lsm 710, by Zeiss (3 mentions)
Ab150157, by Abcam (2 mentions)
Ab176753, by Abcam (2 mentions)
H3570, by Thermo Fisher Scientific (2 mentions)
Ab54210, by Abcam (2 mentions)
Ab150117, by Abcam (2 mentions)
Ab150135, by Abcam (2 mentions)
Ab16663, by Abcam (2 mentions)
Confocal microscopy, by Leica (2 mentions)
Ab191073, by Abcam (2 mentions)
A1r meta confocal microscope, by Nikon (2 mentions)
Cd7d5, by Novus Biologicals (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
69 protocols using ab150078
1
Immunofluorescence Assay for CD4 and CD8 Cells
2
Immunohistochemistry for Cancer Markers
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Anti-CXCL14 (rabbit IgG; NBP1-31398; Novus, Littleton, CO, USA), anti-CXCL16 (rabbit IgG; ab101404, Abcam, Cambridge, MA, USA), anti-CEA (mouse IgG1, clone II-7, M7072, Dako, Glostrup, Denmark) and FITC-conjugated anti-EpCAM (mouse IgG1, clone BerEP4, F0860; Dako) were used as primary antibodies. Mouse IgG, ready to use (Dako), rabbit IgG ready to use (Dako) and FITC-conjugated mouse IgG (F0313; Dako) were used as negative controls. ImmPRESS micropolymer HRP conjugated anti-rabbit IgG (MP-7401, Vector laboratories, Burlingame, CA, USA), ImmPRESS micropolymer HRP conjugated anti-mouse IgG (MP-7402, Vector laboratories), and Alexa Fluor 555-conjugated goat anti-rabbit IgG (ab150078, Abcam) were used as secondary antibodies. The peroxidase substrate used was DAB (3,3′-diaminobenzidine, Vector Laboratories).
3
Immunocytochemical Analysis of Methamphetamine-Induced Neurotoxicity
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VMDNs were fixed in culture using 4% paraformaldehyde after 3 days of methamphetamine treatment (and control cultures) and stored at 4°C in 1× PBS until the staining procedure. TUJ1 (1:1500; Promega) and TH (1:500; Abcam, Cambridge, UK) primary antibodies were incubated with fixed cultures overnight at room temperature in a blocking buffer comprising 5% goat serum, 0.3% Triton-X, and 0.2% sodium azide.
After removing the primary antibodies, the cells were treated with a blocking solution for 1 h at room temperature. Subsequently, the cells were incubated with goat anti-rabbit IgG H&L (Alexa Fluor® 555) and anti-mouse Alexa 488 (Abcam, ab150078) secondary antibodies at 1:200 dilutions for 2 h at room temperature. The wells were then cleaned and maintained in 1× PBS and treated with DAPI (Thermo Fisher Scientific; D1306) diluted in 1× PBS for 5 min. Imaging was performed using a DMi8 inverted fluorescence microscope (Leica, Wetzlar, Germany).
After removing the primary antibodies, the cells were treated with a blocking solution for 1 h at room temperature. Subsequently, the cells were incubated with goat anti-rabbit IgG H&L (Alexa Fluor® 555) and anti-mouse Alexa 488 (Abcam, ab150078) secondary antibodies at 1:200 dilutions for 2 h at room temperature. The wells were then cleaned and maintained in 1× PBS and treated with DAPI (Thermo Fisher Scientific; D1306) diluted in 1× PBS for 5 min. Imaging was performed using a DMi8 inverted fluorescence microscope (Leica, Wetzlar, Germany).
4
Immunofluorescence Staining of Neural Cells
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The cells were fixed with 4% paraformaldehyde (PFA, Solarbio, China) for 15 min, washed with PBS, permeabilized with 0.5% Triton X-100 for 30 min, and blocked with 5% goat serum (Solarbio, China) for 1 h at room temperature. Then, the cells were incubated with the primary antibodies in blocking buffer overnight at 4°C. After washing three times with PBS, the cells were incubated with the fluorescence-labeled secondary antibodies Alexa Fluor® 555 (1:500, ab150078, abcam), Alexa Fluor® 488 (1:500, ab150113, abcam) or CoraLite® 488 (1:500, SA00013-2, Proteintech) for 2 h at room temperature in the dark. A staining solution consisting of 4',6-diamidino-2-phenylindole (DAPI, Solarbio, China) was added to stain the nuclei, and the images were collected under a fluorescence microscope (Olympus, Japan). For cell immunofluorescence staining counts, the number of samples per group was n=5, and 5 fields of view were randomly captured for each sample. The primary antibodies used in this study were as follows: Nestin (1:200, PA5-118114, Invitrogen), GAP-43 (1:500, ab16053, abcam), Sox-2 (1:300, 11064-1-AP, Proteintech), GFAP (1:500, ab10062, abcam), βIII-tubulin (1:500, 66375-1-Ig, Proteintech), Iba-1 (1:500, 019-19741, Wako), NLRP3 (1:200, YT5382, Immunoway), and Caspase-1 (1:300, YT5743, Immunoway).
5
Immunocytochemistry of APPL in MSCs
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The medium in osteogenic differentiated MSCs was removed and washed three times with PBS. Cells were fixed with 4% paraformaldehyde for 30 min and permeated with 0.5% Triton X-100 (Macklin, T824275) for 30 min, and 5% normal goat serum (Solarbio, SL038) was used to block cells for 30 min at room temperature. Then, an appropriate amount of immunostaining primary antibody dilution buffer (Beyotime, P0103) and primary antibody against APPL (Abcam, ab180140, 1:250) were added and incubated overnight at 4 °C. After washing with PBS three times, goat anti-rabbit IgG H&L (Alexa Fluor 555) (Abcam, ab150078, 1:1000) and/or goat anti-mouse IgG H&L (Alexa Fluor 488) (Abcam, ab150113, 1:1000) was added and incubated for 60 min at room temperature. Thereafter, the nuclei of MSCs were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Beyotime, C1006). Finally, the images were observed and collected under a fluorescence microscope (Leica DMI4000 B).
6
Multiplex Immunofluorescence Staining Protocol
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Prepared sections were rehydrated in PBS, boiled in 10 mM citric acid buffer (pH 6.0) at 95 °C for 10 min to unmask antigens, cooled down at room temperature for 30 min, washed with PBS, and permeabilized with 0.1% Triton-X (Sigma-Aldrich, #9002-93-1) in PBS for 5 min. Next, the sections were blocked with 5% donkey serum in PBS for 1 h and incubated with a cocktail primary antibodies in blocking buffer for overnight at 4 °C. After washing with PBS, a cocktail of fluorescence-conjugated secondary antibodies (Goat anti-rabbit-IgG Alexa Flour555-conjugated, Abcam, ab150078; Goat anti-mouse-IgG Alexa Flour488-conjugated, Abcam, an150114; Goat anti-mouse-IgG Alexa Fluor594-conjugated, Thermo Fisher, A-11005; Goat anti-rabbit-IgG FITC-conjugated, Abcam, ab6717) were allowed to bind for 1 h at room temperature. After washing with PBS, the sections were mounted using antifade mounting solution with DAPI (Cell Signaling, #8961) and images were captured using a confocal laser scanning microscope (LSM700; Carl Zeiss).
7
Ileum Immunofluorescence for MD2 and Occludin
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For immunofluorescence, sections of paraffin ileum tissue (4 mm) were prepared and fixed with 4% PFA. Then, 5% goat serum was added for 1 hour at room temperature, and the primary antibodies were applied overnight in a dark chamber at 4°C. After incubating with the second antibody at room temperature for 1 hour, the sections were mounted with VECTASHIELD Antifade Mounting Medium with DAPI to stain the nucleus. Using a Leica SP8 inverted fluorescence microscope, immunofluorescent images were acquired. ImageJ software was used to calculate the average fluorescence intensity of MD2 and occludin.
The following was the primary antibodies and their concentrations: rabbit anti-rat MD2 (ab24182, Abcam) (1:50), mouse anti-rat occludin (66378-1-Ig, Proteintech) (1:200). The secondary antibodies and their concentrations were as follows: goat anti-mouse or rabbit IgG H&L-Alexa Fluor 555 (ab150114 or ab150078, Abcam) (1:1000).
The following was the primary antibodies and their concentrations: rabbit anti-rat MD2 (ab24182, Abcam) (1:50), mouse anti-rat occludin (66378-1-Ig, Proteintech) (1:200). The secondary antibodies and their concentrations were as follows: goat anti-mouse or rabbit IgG H&L-Alexa Fluor 555 (ab150114 or ab150078, Abcam) (1:1000).
8
Immunofluorescence Staining Protocol
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Cells were harvested for fixation in 4% formaldehyde and permeabilisation in .3% Triton X‐100, and then blocked with 2% bovine serum albumin (BSA). After incubating with the primary antibody overnight and goat anti‐mouse IgG H+L (Alexa Fluor 488) (1:300, ab150013, Abcam) or goat anti‐rabbit IgG H+L (Alexa Fluor 555) (1:300, ab150078, Abcam) secondary antibody at 37°C for 30 min, 4,6‐diamino‐2‐phenyl indole (DAPI) (28718‐90‐3, Sigma–Aldrich) was used for staining nuclei for 5 min. Subsequently, the slices were coated with an anti‐quenching reagent, and fluorescent signals were evaluated using a fluorescence microscope (Leica).
9
Immunohistochemistry of mCherry-labeled Retina
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Injected L7Cre mice were culled by cervical dislocation (approved schedule 1 method). Eyes were removed and placed in 4% paraformaldehyde in phosphate‐buffered saline for 24 h at 4°C. Retinas were then dissected and permeabilised in 1% Triton X in PBS for 3 × 10 min at room temperature. Retinas were then blocked using 1% Triton X in PBS with 10% normal goat serum for 2–3 h while shaking gently. Retinas were incubated in primary antibody (1:500 dilution of rabbit anti‐mCherry antibody, Kerafast, catalogue no. EMU106, in 1% Triton X in PBS with 2.5% goat serum) overnight at room temperature. Retinas were washed in PBS with 0.2% Triton X for 4 × 30 min shaking gently. Retinas were incubated in secondary antibody (Goat anti‐rabbit Alexa fluorophore 555, Abcam, catalogue no. ab150078, in 1% Triton X in PBS with 2.5% goat serum) for 3–4 h at room temperature in the dark. Retinas were washed a further four times in PBS with 0.2% Triton X for 30 min each and then washed in ddH20 for 10 min. Retina was then mounted on a microscope slide using Prolong Gold antifade mountant and allowed to dry overnight at room temperature.
10
Klotho and CCL5 Expression Analysis
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Dewaxed kidney tissue sections were rehydrated, heated for 20 min in a microwave for antigen retrieval, and then incubated with immunohistochemical serum blocking agent (GEPbio, 317615) for 30 min at 37°C to block non-specific staining. Subsequently, the sections were incubated with a rabbit anti-Klotho antibody (ab181373, Abcam) and rat anti-CCL5 antibody (NB120-10394, NOVUS) overnight at 4°C. The next day, the sections were washed with PBS for three times and then incubated with an Alexa Fluor 555-labeled anti-rabbit second antibody (ab150078, Abcam) and Alexa Fluor 488-labeled anti-rat second antibody (ab150157, Abcam) at 37°C for 30 min. Then, cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, GEPbio, 721621) for 3 min. Finally, the sections were washed with PBS again and mounted with anti-fade medium (GEPbio, 717615). Stained sections were examined and imaged under a fluorescence microscope. Mean fluorescence intensity (MFI) of each section was analyzed by Image J software.
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