The microglial BV-2 cell line was seeded at 1.2 x 104 density on coverslip properly lodge in 6 multiwells in their complete growth medium. The following day, the medium was replaced with starvation medium supplemented with appropriate stimuli. After each treatment, the medium was discarded and a previously used standard procedure was followed.12 The primary antibody (rabbit anti-cytochrome C, rabbit anti-Iba1, rabbit anti-Nrf2, or mouse anti-CD86; SantaCruz Biotechnology, Santa Cruz, CA, USA), diluted 1:200 in blocking solution were used. The images were acquired by a motorized 654 Leica DM6000 B microscope equipped with a DFC350FX camera (Leica, Mannheim, Germany).
Mouse anti cd86
Manufactured by Santa Cruz Biotechnology
Sourced in United States, China
Mouse anti-CD86 is a monoclonal antibody that binds to the CD86 surface protein, also known as B7-2. CD86 is a costimulatory molecule expressed on the surface of antigen-presenting cells and plays a crucial role in T cell activation and the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti cd206, by R&D Systems (2 mentions)
Rabbit anti cd206, by Cell Signaling Technology (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Dfc350 fx camera, by Leica (1 mentions)
Rabbit anti nrf2, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti cytochrome c, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti iba 1, by Santa Cruz Biotechnology (1 mentions)
Goat serum, by Bioss Antibodies (1 mentions)
Immunofluorescence microscope, by Zeiss (1 mentions)
Rabbit anti cd163, by Bioss Antibodies (1 mentions)
4 6 diamino 2 phenylindole dapi, by Beyotime (1 mentions)
Ab64693, by Abcam (1 mentions)
Alexa 488, by Santa Cruz Biotechnology (1 mentions)
Epr19518, by Abcam (1 mentions)
Rabbit anti inos, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Rabbit anti cd206, by Abcam (1 mentions)
Rabbit anti iba1, by GeneTex (1 mentions)
9 protocols using mouse anti cd86
1
Microglial BV-2 Cell Immunocytochemistry
2
Immunofluorescent Staining of Atrial Tissue
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The frozen sections of atrial tissue were fixed with 4% paraformaldehyde for 15 minutes, washed with 1×PBS for three times, and permeabilized in 1% TritonX-100 for 10 minutes. After washing, the sections were blocked with goat serum (Bioss, Beijing, China) for 1 hour and incubated with primary antibody, rabbit anti-CD163 (Bioss, cat#bs-2527R, RRID: AB_10856166) and mouse anti-CD86 (Santa Cruz Biotechnology, cat#sc-28347, RRID: AB_627200) at 4°C overnight. Following that, they were incubated with FITC Goat Anti-Rabbit IgG (H+L) antibody(ABclonal, cat#AS011, RRID: AB_2769476), and TRITC Goat Anti-Mouse IgG (H+L) antibody (ABclonal, cat#AS026, RRID: AB_2772721). Finally, 4′6-diamino-2-phenylindole (DAPI, Beyotime, China) was added to stain the nuclei. The sections were sealed with anti-fluorescence quencher. Imaging was performed by immunofluorescence microscope (Zeiss, Jena, Germany) and fluorescence intensity was analyzed with ImageJ.
3
Immunofluorescence Staining of Meningioma
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Immunofluorescence staining was performed as described previously.17 (link) Briefly, 4 µm tissue sections were cut in series from formalin-fixed paraffin-embedded tissue blocks for each meningioma case. Standard H&E staining was performed for each tumor and histological features of each section were reviewed by a neuropathologist establishing the WHO grade (Neuropathology report). For immunofluorescence, sections were stained overnight with primary antibodies; mouse anti-CD68 antibodies (1:200, (KP1) conjugated to Alexa-488 Santa Cruz Biotechnology), mouse anti-CD80 (1:100, (R&D Systems), mouse anti-CD86 (1;100, D-6 Santa Cruz Biotechnology together with either rabbit anti-CD163 (1:200, EPR19518, Abcam), rabbit anti-CD206 (1:1000, ab64693, Abcam), or rabbit anti-iNOS (1:200, Abcam). Secondary goat anti-rabbit or anti-mouse Alexa-555 & -488 antibodies (Molecular probes) were applied to sections and tissue auto-fluorescence was blocked with 30-min incubation in 0.02% Sudan black in 70% EtOH. Sections were then coverslipped using hard-set mounting media containing DAPI (Vectashield).
4
Immunofluorescence Staining of Brain Sections
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After washed with PBS (5 min each). Prepared brain sections were permeated with 0.5% Triton X-100 for 30 min, and blocked with 10% goat serum for 2 h at room temperature, followed by overnight incubation with primary antibody at 4°C. The next day, samples were incubated with the corresponding secondary antibody at room temperature for 2 h. And 4′, 6-diamino-2-phenylindole (DAPI; Boster, Wuhan, China) were counterstained for 5 min. Finally, stained sections were examined, and images were captured using a confocal microscope (LSM-880; Zeiss). The primary antibodies used in the experiment are as follows: goat anti-ionized calcium-binding adaptor molecule (Iba1; 1:200; Abcam, UK), rabbit anti-Iba1 (1:200; GeneTex, Irvine, CA, USA), mouse anti-CD86 (1:200; Santa Cruz, CA, USA), rabbit anti-CD206 (1:200; Abcam, UK). Images from four sections of the brain around the PAG, VTA and VPL were captured using a 20× objective on a Zeiss confocal microscope (Zeiss, LSM780, Germany). Cell numbers were calculated per random microscopic field (100 × magnification). All counts were performed in a blinded fashion.
5
Immunofluorescence Analysis of Microglial Markers
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After treatment, the BV2 cells were fixed for 15 min in 4% paraformaldehyde, permeabilized for 7 min with 0.1% Triton X-100, and then blocked for 30 min with 1% BSA. The cells were incubated for 1 h at RT with mouse anti-CD86 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, sc-28347, 1:250), mouse anti-CD68 (Santa Cruz Biotechnology Inc., sc-20060; 1:250), a rabbit anti-IL-10 antibody (Abbiotec, 250713; 1:200), a rabbit anti-TNF-α antibody (Novus Biologicals, NB600-587; 1:100), or a rabbit anti-α7 nAchR (Abcam, ab216485; 1:500). After washing in PBS three times for 5 min each, the cells were incubated for 1 h at RT in the dark with the appropriate fluorescent-labelled secondary antibodies (Alexa Fluor 488 donkey anti-mouse (Thermo Fisher Scientific), Alexa Fluor 546 donkey anti-rabbit (Thermo Fisher Scientific), or Alexa Fluor 488 donkey anti-rabbit (Thermo Fisher Scientific)). Finally, for nuclear staining and the stabilization of fluorescent signals, the slides were covered in mounting medium (Fluoroshield with DAPI; Sigma-Aldrich, Milan, Italy) and secured with a coverslip. Fluorescence images were captured using a Zeiss Observer.Z1 microscope equipped with the Apotome.2 acquisition system (Zeiss LSM 700, Jena, Germany).
6
Immunofluorescence Staining of Brain Slices
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Brain slices were prepared as previously described,
21 (link) and 0.3% Triton ×100 was used to penetrate 15 min. Brain slices (30 μm) were blocked with QuickBlock™ (Beyotime, China) for 1 h, and then incubated in a refrigerator with primary antibodies at 4°C overnight. We performed single immunofluorescence staining using rabbit anti‐Iba1 (1:1000, Abcam, USA). Performed double immunofluorescence staining using mouse anti‐Iba1 (1:100, Santa Cruz Biotechnology, USA) with rabbit anti‐TRPV4 (1:200, Abcam, USA), rabbit anti‐Iba1 (1:1000, Abcam, USA) with mouse anti‐CD86 (1:50, Santa Cruz Biotechnology, USA), and rabbit anti‐Iba1 (1:1000, Abcam, USA) with goat anti‐CD206 (1:250, R&D Systems, USA). After washing the brain slices with PBS containing 0.3% Triton ×100, Alexa 594‐conjugated goat anti‐rabbit antibodies (1:1000, Life Technologies Corporation, USA), Alexa 555‐conjugated donkey anti‐goat antibodies (1:1000, Life Technologies Corporation, USA), and Alexa 488‐conjugated goat anti‐rabbit antibodies (1:1000, Life Technologies Corporation, USA) were added and incubated at room temperature for 2 h. The nucleus was stained with DAPI. Using laser scanning confocal microscopy (Leica SP‐8) to take pictures. ImageJ was used for analysis.
21 (link) and 0.3% Triton ×100 was used to penetrate 15 min. Brain slices (30 μm) were blocked with QuickBlock™ (Beyotime, China) for 1 h, and then incubated in a refrigerator with primary antibodies at 4°C overnight. We performed single immunofluorescence staining using rabbit anti‐Iba1 (1:1000, Abcam, USA). Performed double immunofluorescence staining using mouse anti‐Iba1 (1:100, Santa Cruz Biotechnology, USA) with rabbit anti‐TRPV4 (1:200, Abcam, USA), rabbit anti‐Iba1 (1:1000, Abcam, USA) with mouse anti‐CD86 (1:50, Santa Cruz Biotechnology, USA), and rabbit anti‐Iba1 (1:1000, Abcam, USA) with goat anti‐CD206 (1:250, R&D Systems, USA). After washing the brain slices with PBS containing 0.3% Triton ×100, Alexa 594‐conjugated goat anti‐rabbit antibodies (1:1000, Life Technologies Corporation, USA), Alexa 555‐conjugated donkey anti‐goat antibodies (1:1000, Life Technologies Corporation, USA), and Alexa 488‐conjugated goat anti‐rabbit antibodies (1:1000, Life Technologies Corporation, USA) were added and incubated at room temperature for 2 h. The nucleus was stained with DAPI. Using laser scanning confocal microscopy (Leica SP‐8) to take pictures. ImageJ was used for analysis.
7
Western Blot Analysis of Neuroinflammation Markers
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The cerebral cortex tissue was dissolved in RIPA buffer (Beyotime, Nantong, China), which contained an inhibitor. The tissues were ground, sonicated, and then centrifuged to obtain supernatants. Total protein expression was quantified using the BCA kit (Beyotime, Nantong, China). Subsequently, protein samples were electrophoresed and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% bovine serum albumin for 1.5 hours, the membranes were incubated overnight with primary antibodies. The primary antibodies included rabbit anti-TLR4 (1:1000; Abcam, USA), rabbit anti-TIR-domain-containing adaptor inducing interferon-β (TRIF) (1:1000; Proteintech, China), mouse anti-CD86 (1:500; Santa Cruz, Europe), rabbit anti-CD206 (1:1000; Cell Signaling Technology, USA), mouse anti-Iba-1 (1:1000; Synaptic Systems, Germany), and mouse anti-β-actin (1:2000; Proteintech, China). Following primary antibody incubation, the membranes were incubated for 2 hours with the appropriate horseradish peroxidase-conjugated secondary antibody. The protein bands were quantified using ImageJ 1.8.0 in the presence of chemiluminescence.
8
Evaluating NF-κB p65 Expression in RAW264.7 Cells
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Immunofluorescence staining of RAW264.7 cells was carried out to evaluate the level of protein expression and the distribution of NF-κB p65. We seeded 1 × 10 3 cells per well in 24-well plates containing glass coverslips. The test cells were subjected to treatment with GLPS for 24 h. After the treatment and removal of medium, we fixed the cells with 4% paraformaldehyde for 15 min and then permeabilized them in 0.1% Triton X-100 (Solarbio, China) for 10 min. Subsequently, nonspecific binding sites were blocked at RT for an hour in 5% bovine serum albumin (BSA) buffer (Solarbio, China). After rinsing three times with PBST (PBS containing 0.1% Tween-20), the cells were incubated with mouse anti-CD86 (diluted 1 : 200, Santa Cruz, USA), goat anti-CD206 (diluted 1 : 200, RD, USA), and rabbit anti-P65 (diluted 1 : 200, Abcam, UK) overnight at 4 degrees Celsius. Then, we stained the cells with the corresponding secondary FITC-conjugated antibodies (diluted 1 : 200, Proteintech, USA) or PE-conjugated antibodies (diluted 1 : 200, Proteintech, USA) at RT for an hour in the dark.
Finally, an antifade mounting medium containing DAPI (Solarbio, China) to visualize the nucleus was dripped on the glass slides. The slides were stained for 5 minutes at RT. A fluorescence microscope (IX81, Olympus, Japan) was used to observe and photograph the labeled cells.
Finally, an antifade mounting medium containing DAPI (Solarbio, China) to visualize the nucleus was dripped on the glass slides. The slides were stained for 5 minutes at RT. A fluorescence microscope (IX81, Olympus, Japan) was used to observe and photograph the labeled cells.
9
Microglial Phenotype Analysis in Rat Brain Slices
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Rat brains were sectioned, and the resulting slices were washed three times in 0.1 M PBS for subsequent experiments. The slices were then incubated with 0.3% Triton X-100 for 20 minutes, followed by another three washes with 0.1 M PBS. After a 1-hour incubation in 2% donkey serum albumin, the slices were subjected to overnight incubation at 4°C with the respective primary antibodies: Guinea pig anti-Iba-1 (1:500; Synaptic Systems, Germany), mouse anti-CD86 (1:100; Santa Cruz, USA), and rabbit anti-CD206 (1:500; Cell Signaling Technology, USA). Nuclei were labeled using 4’6-diaminoamino-2-phenylindole dihydrochloride (DAPI). To visualize the target proteins, the sections were incubated with Alexa-488 (green) or Alexa-594 (red) conjugated donkey anti-rabbit or donkey anti-mouse secondary antibodies for 2 hours. Changes in microglial morphology and the numbers of Iba-1, CD86, and CD206 positive cells were tracked using confocal laser scanning microscopy.
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