Immunofluorescence (IF) staining was performed as previously described but with some modifications (Chen et al., 2015 (link)). Briefly, coronal paraffin-embedded 4-μm thickness brain sections were prepared as mentioned above. The sections underwent antigen retrieval and were then blocked in 5% BSA for 1 h. After blocking, sections were incubated overnight at 4°C with the following primary antibodies used: rabbit anti-GSDMD (1:200, Ab219800, Abcam), rabbit anti-caspase-1 (1:200, Ab238972, Abcam), rabbit anti-p-AKT (1:200, 4,060, Cell Signaling Technology), rabbit ani-p-GSK3β (1:200, 5,558, Cell Signaling Technology), and mouse anti-Iba1 (1:200; GB12105, Servicebio). The next day, the slices were washed with PBS and incubated with secondary antibodies: donkey anti-goat Alexa 488 (1:500, A11055, Invitrogen) and donkey anti-rabbit Alexa 555 (1:500, A31572, Invitrogen) for 1 h at room temperature. After washing three times with PBS, the sections were re-stained by 4′6-diamidino-2-phenylindole (DAPI) for 10 min. Then, images were acquired using a fluorescence microscope (ZEISS-AXIO Scope. Al, Germany).
Gb12105
Manufactured by Wuhan Servicebio Technology
The GB12105 is a laboratory equipment product. It is designed for conducting specific laboratory tasks. The core function of this product is to provide a controlled environment or to perform specific operations required in a laboratory setting. A detailed description of the product's features and intended use is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab219800, by Abcam (3 mentions)
Gb21303, by Wuhan Servicebio Technology (3 mentions)
Gb12096, by Wuhan Servicebio Technology (2 mentions)
Gb25301, by Wuhan Servicebio Technology (2 mentions)
Cy3 labelled secondary antibody, by Wuhan Servicebio Technology (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Af4005, by Affinity Biosciences (2 mentions)
Ab238972, by Abcam (1 mentions)
A31572, by Thermo Fisher Scientific (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
Rabbit anti p akt, by Cell Signaling Technology (1 mentions)
Ab150108, by Abcam (1 mentions)
Ab243074, by Abcam (1 mentions)
Ds u3, by Nikon (1 mentions)
Bs114, by Biosharp (1 mentions)
G1012, by Wuhan Servicebio Technology (1 mentions)
Eclipse c1, by Nikon (1 mentions)
Ag 20b 0014 c100, by Adipogen (1 mentions)
Ab22048, by Abcam (1 mentions)
Ab6671, by Abcam (1 mentions)
12 protocols using gb12105
1
Immunofluorescence Staining of Brain Sections
2
Histological Analysis of Spinal Cord
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The spinal cords were fixed with 4% paraformaldehyde (PFA), embedded in paraffin, and cut into slices of 4 μm thickness. After deparaffinization and rehydration, hematoxylin-eosin (HE) and Luxol Fast Blue (LFB) staining were performed as previously described [19 (link)]. For fluorescence immunohistochemistry, the sections were incubated overnight with mouse monoclonal primary antibody against IBA1 (Servicebio, GB12105, 1:150) at 4 °C, followed by staining with a donkey anti-mouse IgG secondary antibody (Abcam, ab150108, 1:200). For fluorescence immunocytochemistry, the cells were harvested after gently repeated blowing, centrifuged at 1000 rpm, fixed with 4% PFA, permeabilized with 0.1% Triton ×100, and incubated with primary mouse monoclonal anti-MGO antibody (Abcam, ab243074, 1:200) at 4 °C overnight, followed by staining with donkey anti-mouse IgG secondary antibody (Abcam, ab150108, 1:200). All images were captured using Olympus BX51 or LEICA DMi8 microscope.
3
Immunofluorescence Localization of Stress Sensors
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The localization of PERK, STING, p-TBK1, and p-IRF3 were identified by immunofluorescence. Paraffin sections of brain tissue were dewaxed with xylene and dehydrated with gradient alcohol. Antigen repair was performed with EDTA (pH 8) and heated in the microwave. After cooling naturally, the sections were washed thrice with phosphate-buffered saline (PBS, pH 7.4) and blocked using 5% bovine serum albumin (BSA; #BS114, Biosharp) for 1 h at room temperature. Subsequently, slices were incubated overnight at 4°C with mouse anti-GFAP (glial fibrillary acidic protein, #GB12096,1:500, Servicebio) to identify astrocytes, mouse anti-ionized calcium-binding adaptor molecule 1 (Iba-1; #GB12105,1:500, Servicebio) to identify microglia, mouse anti-NeuN (Neuronal Nuclei, #K009907M,1:200, solarbio) and primary antibody against PERK (1:50), STING (1:100), p-TBK1(1:100), and p-IRF3 (1:200), respectively. After three washes with PBS on the next day, slices were incubated in Cy3-conjugated anti-rabbit (GB21303,1:200, Servicebio) and AlexaFluor488-conjugated anti-mouse (GB25301,1:400, Servicebio) secondary antibodies for 50 min in the dark. Finally, DAPI (G1012, Servicebio) was added to the sections for 15 min to identify the nuclei. Images were acquired on an inverted fluorescence microscope (Eclipse C1; Nikon) at 40× objective and an imaging system (DS-U3; Nikon).
4
Immunofluorescent Staining of Brain Tissue
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Immunofluorescent stain was performed on paraffin-embedded sections or frozen sections of brain tissue. Paraffin sections were dewaxed and hydrated and underwent antigen retrieval, but frozen sections did not require this processing. The sections were treated with 5% bovine serum albumin and sealed at room temperature for 30 min. Then, diluted primary antibody was added to each section and incubated overnight in a wet box at 4 °C. After the excess primary antibody was washed away, the diluted fluorescent secondary antibody was added to the brain slice and incubated at 20–37 °C for 1 h in the dark. 4ʹ,6-diamino-2-phenylindole (DAPI) staining was performed for 5–10 min to label the nuclei. The sections were observed with an Olympus Automatic Scanning System SV120. The following primary antibodies were used: rabbit anti-IBA1 (NO. 019-19741, Wako), rabbit anti-iNOS (18985-1-AP, Proteintech), mouse anti-Arg-1 (66129-1-Ig, Proteintech), mouse anti-IBA1 (GB12105, Servicebio), and mouse anti-NLRP3 (AG-20B-0014-C100, Adipogen).
5
Immunofluorescence Analysis of TLR4 and TNF-α
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Immunofluorescence colocalization analysis was performed to examine the expression of TLR4 and TNF-α in brain cells and the expression of TNF-α in microglia. The paraffin-embedded 4-μm sections were deparaffinized and rehydrated before antigen retrieval by microwaving. Following the application of blocking serum, the sections were incubated with the following primary antibodies overnight at 4°C: anti-TLR4 antibody (1:100, ab22048, Abcam), anti-TNF-α antibody (1:100, ab6671, Abcam), anti-IBA-1 antibody (1:500, GB12105, Servicebio) and anti-TNF-α antibody (1:100, ab6671, Abcam). The sections were then incubated with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (1:400, GB25301, Servicebio) and CY3-conjugated goat anti-rabbit secondary antibody (1:300, GB21303, Servicebio) for 50 min at room temperature, rinsed, counterstained with DAPI and air-dried before mounting. Fluorescence was imaged by fluorescence microscope. The number of TNF-α/TLR-4 and TNF-α/IBA-1 co-positively immunostained cells in each section of identical regions of interest was determined in eight high-power fields (HPFs; 400× magnification) and defined as cell density (cells/HPF).
6
Immunohistochemical Analysis of NLRP3 and Microglia
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IHC was used to verify NLRP3 and IBA1 (a microglial marker in the brain) expression in the ipsilateral cortex. The deparaffinized and rehydrated coronal brain slices (4 μm thick) were prepared as described above. The slices were incubated with 3% H2O2 for 10 min at room temperature to quench any endogenous peroxidase activity, and then blocked with 5% goat serum for 20 min at room temperature. The slices were then incubated overnight at 4°C with the following primary antibodies: rabbit anti-NLRP3 (diluted 1:50; NBP1-77080SS, Novus) and mouse anti-IBA1 (diluted 1:100; GB12105, Servicebio). The next day, the brain slices were washed with PBS and incubated for 20 min at room temperature with biotinylated goat anti-rabbit IgG (diluted 1:100; ZSGB-Bio, Beijing, China) or goat anti-mouse IgG (diluted 1:100; ZSGB-Bio). The brain slices were then incubated with horseradish peroxidase (HRP)–streptavidin reagent for 10 min and stained with 3,3′-diaminobenzidine peroxidase substrate. Images were obtained with a light microscope (Leica-DM2500, Wetzlar).
7
Microglial Morphology Analysis in Hypothalamic PVN
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At the end of the experiment, hypothalamic PVN tissues of rats in each group were obtained for immunofluorescence staining. Tissues were sectioned into 5-μm slices and blocked with 5% normal fetal bovine serum in PBS for 2 h. Thereafter, the slices were incubated with primary antibody against Iba-1 (1:200, GB12105, Servicebio) overnight at 4 °C. The slices were then rinsed with PBS and incubated with Cy3-labelled secondary antibody (1:300, Servicebio) in the dark at room temperature for 60 min. To determine the microglial morphology, the staining imaging was observed with a fluorescence microscope and handled by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). Three microglial cells per brain slice, three brain slices per animal were measured from the hypothalamic PVN. The parameters of microglial morphology include soma area, processes length, and the number of primary branch projection (ramification). The number of Iba-1-positive cells was also calculated.
8
Immunohistochemical Analysis of PVN Markers
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At the end of the experiment, the PVN tissues of all rats were obtained, sectioned into 5 μm slices, and blocked with 5% bovine serum albumin for 30 minutes at room temperature. They were then incubated overnight with primary antibody Iba-1 (1 : 200, GB12105, Servicebio) or BDNF (1 : 200; Google Bio#GB11240) or CD206 (1 : 200; Google Bio#GB11062) in a wet box at 4°C. Afterwards, sections were rinsed with PBS and incubated with the corresponding Cy3-labelled secondary antibody (1 : 300, Servicebio) in the dark for 50 minutes at room temperature. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (G1012, Servicebio). Images were captured with a fluorescence microscope and handled by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). The positive area of Iba-1-positive, BDNF, and CD206 cells was counted in 3 serial sections at 400× magnification. The average of the 3 serial sections was taken. Same method was used to measure the area of DAPI.
9
Immunohistochemical Analysis of Spinal Cord Injury
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On postoperative day 7, the L4–L6 spinal cord was harvested from rats (n = 3 for SCI group), embedded in paraffin, and cut into 20-μm-thick sections. These sections (n = 3 for each sample) were dewaxed, dehydrated by gradient alcohol, and repaired by antigen. Next, the sections were blocked with endogenous peroxidase and 10% donkey serum and then incubated with the following primary antibodies: NLRP3 (1:200; bs-6655R, Bioss), GFAP (1:500; GB12096, Servicebio), IBA-1(1:500; GB12105, Servicebio), and NeuN (1:100; GB13138-1, Servicebio) overnight at 4°C. After the primary antibody incubation, the sections were incubated with the corresponding secondary antibodies conjugated with CY3 and FITC for 1 h in dark conditions at 37°C. Finally, the dorsal horns were observed and photographed under a fluorescence microscope (Olympus, Japan).
10
Immunofluorescence Imaging of Microglia Markers
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Sections of the prefrontal cortex (PFC) were incubated with primary antibodies overnight and then secondary antibodies in the dark for 1 h. The antibodies used for IF staining were as follows: anti-IBA-1 (1:200, GB12105, Servicebio), anti-CD68 (1:100, GB113109, Servicebio), anti-CR3 (1:500, GB11058, Servicebio), CY3 goat anti-mouse secondary antibody (1:300, GB21303, Servicebio), and 448 AffiniPure Fab Fragment goat anti-rabbit secondary antibody (1:500, GB25303, Servicebio). The brain slices in our study underwent a blocking step using 10% goat normal serum [29 (link)]. Samples were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for 10 min and then visualized using a fluorescence microscope (Nikon Eclipse C1, Japan).
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