The cultures were fixed in 4% paraformaldehyde (PFA) for 20 min and were washed with phosphate‐buffered saline (PBS) before blocking nonspecific binding and permeabilizing the cells with 5% bovine serum albumin (BSA) and 5% donkey serum plus 0.1% Triton X‐100 (Sigma, Dorset, UK) in PBS for 1 hr at room temperature. Neurons were incubated overnight with the primary antibody in 1% blocking solution at 4°C. After washing with PBS, the cultures were incubated with the appropriate secondary antibody. The primary antibodies were: anti‐class III β tubulin (1:500, Abcam, ab41489), anti‐CD40 (1:500, MA5‐15535, Invitrogen), and anti‐CD40L (1:200, Abcam, ab2391). Secondary antibodies were Alexa Fluor conjugated anti‐immunoglobulin from Thermo Fisher Scientific used at 1:500 (donkey anti‐rabbit IgG Alexa Fluor 488, A32790, goat anti‐chicken Alexa Fluor 594, A11042 and goat anti‐mouse Alexa Fluor 488, A11001). Images were obtained using a Zeiss LSM 710 confocal microscope.
Ab2391
Manufactured by Abcam
Sourced in United Kingdom
Ab2391 is a primary antibody that can be used for the detection of a specific target protein in various experimental applications. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
Alexa fluor conjugated anti immunoglobulin, by Thermo Fisher Scientific (1 mentions)
A 11042, by Thermo Fisher Scientific (1 mentions)
Ab13545, by Abcam (1 mentions)
Mab1195, by R&D Systems (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
Axiovert 200 inverted fluorescence microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ab68185, by Abcam (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ab34843, by Abcam (1 mentions)
Ab213987, by Abcam (1 mentions)
Ab272902, by Abcam (1 mentions)
Ab22552, by Abcam (1 mentions)
Ab6994, by Abcam (1 mentions)
Ab18207, by Abcam (1 mentions)
6 protocols using ab2391
1
Immunofluorescence Staining of Neurons
2
Immunofluorescence Staining of CD40 and CD40L
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The culture medium was gently aspirated and the cultures were washed with PBS at 37°C. Cells were fixed with either ice-cold methanol (MeOH) for 5 min or freshly made 4% paraformaldehyde (Sigma-Aldrich) in 0.12 M phosphate buffer (pH 7.2) for 12 min. The fixative was removed and, after washing with PBS, the cultures were blocked for ∼1 h at room temperature in 5% bovine serum albumin (BSA) containing 0.2% Triton X-100 (Sigma-Aldrich). The cultures were then incubated overnight at 4°C with primary antibody in PBS containing 1% BSA (Sigma-Aldrich) and were gently agitated on an orbital shaker. After extensive washing in PBS, the cultures were incubated with fluorophore-conjugated secondary antibody (Alexa Fluor 488/546, Thermo Fisher Scientific) in 1% BSA for 1 h in the dark at room temperature. Following serial washes in PBS, the nuclei were counterstained with DAPI (1:10,000, Life Technologies) for 5 min.
The cultures were imaged using either a Zeiss Axiovert 200 Inverted Fluorescence microscope or a Zeiss LSM 710 Confocal Laser Scanning Microscope using Zen software (Zeiss). The following primary antibodies were used: rabbit polyclonal anti-CD40L (1:300, ab2391, Abcam), rabbit polyclonal anti-CD40 (1:300, ab13545, Abcam), mouse monoclonal anti-CD40 (1:300, ab91075, Abcam) and mouse monoclonal anti-βIII-tubulin (1:500, MAB1195, R&D Systems).
The cultures were imaged using either a Zeiss Axiovert 200 Inverted Fluorescence microscope or a Zeiss LSM 710 Confocal Laser Scanning Microscope using Zen software (Zeiss). The following primary antibodies were used: rabbit polyclonal anti-CD40L (1:300, ab2391, Abcam), rabbit polyclonal anti-CD40 (1:300, ab13545, Abcam), mouse monoclonal anti-CD40 (1:300, ab91075, Abcam) and mouse monoclonal anti-βIII-tubulin (1:500, MAB1195, R&D Systems).
3
CD40L and 41BBL Expression in Ovarian Cancer Xenograft
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Example 2
This example describes in vivo expression of CD40L and 41BBL in a human SKOV3 ovarian cancer xenogeneic model.
6-8 week old nude mice were administered with 4.5×106 SKOV3.Luc cells. Once tumors reached in average 150 mm3, mice were injected intratumorally (i.t.) with 1×1010 v.p. per mouse or the same volume of PBS (30 μl).
Twenty four hours later, tumors were removed and protein extracts were prepared. One hundred μg of protein extracts were separated in 12% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories). The membranes were probed with anti-h4-1-BBL antibody (ab68185 Abcam, Cambridge, UK) or anti-CD40L antibody (ab2391 Abcam, Cambridge, UK). Anti β-actin antibody (A4700; Sigma, St. Louis, Mo.) and anti-α tubulin (12g10 DSHB) were used as loading controls. Enhanced chemiluminescence reagents were used to detect the signals following the manufacturer's instructions (Amersham, Little Chalfont, UK). CD40L expression is show in
4
Histological Analysis of Tartrate-Resistant Acid Phosphatase
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The tissues were fixed with 4% paraformaldehyde for 2 days, decalcified with a
nitric acid solution for 16–18 h, and subjected to conventional dehydration,
clearing and paraffin embedding, followed by sectioning (3 μm), heating, and
dewaxing and rehydration. The sections were soaked in 3% hydrogen peroxide for
10 min. After being washed in PBS with tween-20 (PBST), the sections were then
incubated with 1:100 diluted rabbit anti-mouse antibody to tartrate-resistant
acid phosphatase (TRAP; ab2391, Abcam, Cambridge, UK) at 4°C overnight. Next,
the sections were incubated with 1:200 diluted HRP-conjugated goat anti-rabbit
antibody for 60 min at room temperature followed by PBST washes. The sections
were then developed with diaminobenzidine for 30 s, rinsed under running water
for 5 min, and stained with hematoxylin for 1 min. Finally, the sections were
dehydrated, cleared, sealed and subjected to microscopic observation.
nitric acid solution for 16–18 h, and subjected to conventional dehydration,
clearing and paraffin embedding, followed by sectioning (3 μm), heating, and
dewaxing and rehydration. The sections were soaked in 3% hydrogen peroxide for
10 min. After being washed in PBS with tween-20 (PBST), the sections were then
incubated with 1:100 diluted rabbit anti-mouse antibody to tartrate-resistant
acid phosphatase (TRAP; ab2391, Abcam, Cambridge, UK) at 4°C overnight. Next,
the sections were incubated with 1:200 diluted HRP-conjugated goat anti-rabbit
antibody for 60 min at room temperature followed by PBST washes. The sections
were then developed with diaminobenzidine for 30 s, rinsed under running water
for 5 min, and stained with hematoxylin for 1 min. Finally, the sections were
dehydrated, cleared, sealed and subjected to microscopic observation.
5
Comprehensive Protein Analysis of Bone Tissue
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Total proteins were extracted from fresh bone tissue with Radio Immunoprecipitation Assay (RIAP) (Beyotime, China) and separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The following specific primary antibodies are used: anti-osteoclast-associated receptor (OSCAR) (ab156742, Abcam, 1:1000), anti-tartrate-resistant acid phosphatase (TRAP) (ab2391, Abcam, 1:1000), anti-HMGB-1 (6893, CST, 1:1000), anti-OSTERIX (ab22552, Abcam, 1:1000), anti-COL1A1 (96321S, CST, 1:1000), anti-DLX2 (ab272902, Abcam, 1:1000), anti-IL-10 (ab34843, Abcam, 1:1000), anti-iNOS (ab213987, Abcam, 1:1000) and anti-β-actin (3700, CST, 1:1000). EnlightTM (Engreen, China) was used for protein band imaging.
6
Immunofluorescent Analysis of Femur Sections
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Femur specimens were dehydrated in 30% sucrose and 10% gum Arabic for 3 days at 4 °C, embedded in optimal cutting temperature compound. Then, 10 μm-thick sections were prepared. Immunofluorescent staining was performed according to standard protocols. Briefly, sections were permeabilized in 0.2% Triton X-100 (Sigma), nonspecific binding was blocked in 10% donkey serum (Solarbio), and sections were incubated with primary antibodies against rat vWF (ab6994, Abcam, 1:200), TRAP (ab2391, Abcam, 1:200), βIII-tubulin (ab18207, Abcam, 1:2000), NF-H (ab8135, Abcam, 1:1000), CYP40 (12716-1-AP, Proteintech, 1:100), MIF (ab7202, Abcam, 1:250), CFL2 (sc-166958, Santa Cruz, 1:200), TPPP3 (sc-244483, Santa Cruz, 1:200), AHR (17840-1-AP, Proteintech, 1:100),,TRPV1 (sc-398417, Santa Cruz, 1:200), Ctsk (sc-48353, Santa Cruz, 1:100), Osx (ab209484, abcam, 1:1000), CD45 (NB100-77417SS, Novus, 1:100) , or Mtsn (sc-13122, Santa Cruz, 1:100) overnight at 4 °C. Fluorescent-conjugated secondary antibodies were used to detect fluorescent signals, followed by counterstaining with Hoechst 33342 (Sigma Aldrich, 1000×). Images of the center field of view were captured for each independent sample using a confocal microscope (A1R, Nikon) and immunofluorescent staining intensity was quantified using Image J software.
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