In brief, the aforementioned brain sections were blocked in goat serum for 15 min, incubated in rabbit anti-CD32 (dilution: 1:50, Santa Cruz Biotechnology, Dallas, TX, USA) and mouse anti-Iba-1 (dilution: 1:300, Abcam, Cambridge, UK) or rabbit anti-CD206 (dilution: 1:200, Proteintech, Wuhan, China) and mouse anti-Iba-1 (dilution: 1:300, Abcam) over night at 4°C, and subjected to Cy3-labeled IgG antibody (dilution: 1:200, Beyotime Institute of Biotechnology, Shanghai, China) and FITC labeled IgG antibody (dilution: 1:200, Beyotime Institute of Biotechnology) for 90 min at room temperature. Finally, 4’, 6-diamidino-2-phenylindole (DAPI, Biosharp, Hefei, China) was used to visualize the nuclei and the target protein was observed under a fluorescence microscope at 400× magnification. Immunofluorescence was quantified by counting the number of co-stained cells. The number of co-stained cells was counted in three random fields.
Mouse anti iba1
Manufactured by Abcam
Sourced in United States, United Kingdom
Mouse anti-Iba1 is a primary antibody that specifically recognizes the Iba1 protein. Iba1 is a calcium-binding protein that is expressed in microglia and plays a role in microglial activation and migration.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti neun, by Abcam (3 mentions)
Mouse anti gfap, by Abcam (3 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Rabbit anti inos, by Abcam (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Donkey anti rabbit igg alexa 488, by Thermo Fisher Scientific (1 mentions)
Rabbit anti p2x4rs, by Alomone (1 mentions)
Alexa fluor 594 igg goat anti rabbit, by Beyotime (1 mentions)
Goat anti ki67, by Abcam (1 mentions)
Alexa fluor 488 goat anti mouse, by Beyotime (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Rabbit anti pcreb, by Cell Signaling Technology (1 mentions)
Rabbit anti bdnf, by Abcam (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Fluoview, by Olympus (1 mentions)
18 protocols using mouse anti iba1
1
Immunofluorescence Analysis of Microglial Markers
2
Immunohistochemistry Protocol for Cell Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cell culture immunohistochemistry, established protocols were employed (Baxter et al., 2011 (link); Puddifoot et al., 2012 (link)). Briefly, cells were fixed in 4% formaldehyde for 20 min at room temperature, washed with PBS and permeabilized with the detergent NP40 (Life Technologies). Cells were subsequently incubated in primary antibody over night at 4°C. The next day, cells were washed with PBS and incubated with the appropriate Alexa Fluor-conjugated secondary antibody (1:250, ThermoFisher) at room temperature for 1h. Cells were then mounted using the mounting medium Vectashield (Vector Labs). Primary antibodies used include: mouse anti-GFAP (1:400, Sigma), mouse anti-Iba1 (1:750, Abcam), rabbit anti-Iba1 (1:1000, Wako Fujifilm), Cy3-conjugated NeuroChrom (1:500, Merck Millipore), mouse anti-Brevican (1:500, Sigma), biotinylated Wisteria Floribunda agglutinin (1:400, Vector Labs). Images were prepared on a Leica AF6000 using a DFC30X digital camera. For morphometric quantification of microglia, cultures were fixed as above and probed for Iba1. Images were taken using a Nikon A1R confocal microscope. Cell outlines were traced and perimeter/area calculated using ImageJ, with 120 cells measured per condition.
3
Immunofluorescence Analysis of Spinal Cord
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
On the 14th day after surgery, the animals were deeply anesthetized with penthiobarbital sodium and transcardially perfused from the ascending aorta with normal saline and 4% paraformaldehyde (PFA) in 0.1 mol/L phosphate buffer (pH 7.4), respectively. After L4–5 spinal cords were removed, post fixed in same fixative for 3 hr, and dehydrated by 30% sucrose in phosphate buffer over two nights. After being cut into 25‐μm thick sections using a cryostat, the transverse spinal sections were processed for immunofluorescence. All the blocked sections were blocked with 5% goat serum in 0.3% Triton X‐100 for 1 hr at room temperature (RT) and incubated with mouse anti‐iba1 (1:500, Abcam) and rabbit anti‐P2X4Rs (1:200, Alomone Labs) over night at 4°C. After three rinses in PBS, all the sections were treated by a mixture of Alexa 647 donkey anti‐mouse IgG and Alexa 488 donkey anti‐rabbit IgG (1:200, Thermo Fisher) for 2 hr at RT, and thereafter, the preparations were washed in PBS. Olympus IX73 (Olympus Optical, Olympus Co. Ltd., Tokyo, Japan) fluorescence microscope was used to analyzed the stained sections, and images were captured with a CCD spot camera. The primary antibodies were omitted during staining when serving as a negative control, and further examined by Western blot.
4
Immunofluorescence Analysis of Neural Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The prepared brain slices were washed with 50 mM PBS for 3 times for 10 min each, and then, the slices were incubated in 3% H2O2 for 30 min to block endogenous peroxidase activity, followed by immersion in blocking solution for 2h and then incubated overnight with primary antibodies: goat anti-Nestin (1:50, Abcam, UK), goat anti-ki67 (1:100, Abcam UK), goat anti-TrkB (1:100, Abcam UK), mouse anti–NeuN (1:200, Abcam UK), mouse anti–GFAP (1:200, Abcam UK), mouse anti–IBA1 (1:200, Abcam UK). Then, the sections were incubated for 2h at 25°C in the dark with the secondary antibodies: Alexa Fluor 488 goat anti-mouse (1:200, Beyotime, China) for TrkB and ki67; Alexa Fluor 594 IgG goat anti–rabbit (1:200, Beyotime, China) for Nestin, GFAP, IBA1 and NeuN. And then the sections were washed by PBS three times, next wiped the surrounding liquid and re-stained nucleus with DAPI. They were observed by the microscope and analyzed using Image-pro Plus 6.0 software (USA).
5
Immunofluorescent Analysis of Rat Brain Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As previously described [40 (link), 41 (link)], rats (n = 8 per group) were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and perfused with PBS followed by fresh 2% glutaraldehyde and 4% paraformaldehyde through the ascending aorta. The brain was collected and fixed in 4% paraformaldehyde for 4 h and then dehydrated in 30% sucrose overnight at 4°C. The brain tissues were embedded in the optimal cutting temperature compound and finally cut to a thickness of 15 μm in a -20°C cryostat for immunofluorescent staining. After blocking in 10% normal goat serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the tissue sections were incubated overnight at 4°C in 10% normal goat serum in PBS containing primary antibodies such as rabbit anti-pCREB, 1 : 150, Cell Signaling Technology; rabbit anti-BDNF, 1 : 100, Abcam; mouse anti-Neun, 1 : 300, Abcam; mouse anti-GFAP, 1 : 500, Abcam; and mouse anti-Iba1, 1 : 500, Abcam. Afterwards, these slices were incubated with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibodies and Alexa Fluor 488-conjugated goat anti-mouse secondary antibodies (1 : 600, Jackson ImmunoResearch) for 1 h. Then, they were washed in PBS and cover-slipped with VECTASHIELD Mounting Medium with DAPI (Cat: H-1200, Vector Lab). Images were captured by a microscopic imaging system (Olympus BX61 and FluoView).
6
Immunofluorescence Analysis of Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The paraffin section (5 μm-thick) of the brain was prepared, after being fixed, washed, permeabilized, and blocked, cells were incubated with rabbit anti-ZO-1 (1:200, Abcam), or rabbit anti-Iba-1 (1:100, Proteintech). After that, sections were further incubated with the fluorescein 488-conjugated anti-rabbit antibody (1:1000, Jackson Immunoresearch). Slides were imaged using a fluorescence microscope. To explore the polarization reprogramming effects of DEX, we performed immunofluorescence double labeling. Sections were incubated in mouse anti-Iba-1 (1:100, Abcam), with rabbit anti-iNOS (1:100, Abcam) or rabbit anti-CD206 (1:100, Abcam) for 1 h at 37°C. Antibodies were then used: the fluorescein 488-conjugated anti-rabbit antibody (1:1000, Jackson Immunoresearch, United States), or Cy3-conjugated anti-mouse antibody (1:1000, Jackson Immunoresearch).
7
Dual Immunofluorescence Analysis of Neural Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Double Immunofluorescence staining was performed on fixed frozen brain sections as previously described.17 (link) The 8-μm thick slides were rinsed with PBS, and permeabilized with 0.3% Triton X-100 for 15min at room temperature. Next, slides were incubated with blocking solution (95% PBS, 5% normal donkey serum, and 0.05% Triton X-100) for 2 hours. Then, the slides were incubated using the following: goat anti-NeuN (neuronal nuclei; 1:300), mouse anti-Iba1 (ionized calcium-binding adaptor molecule 1; 1:300), rabbit anti-FKN (1:100), rabbit anti-CX3CR1 (1:100), rabbit anti-CD68 (cluster of differentiation 68; 1:150), rabbit or goat anti-CD206 (the mannose receptor; 1:200), goat anti-CD206 (1:200), rabbit anti-CD163 (1:100) or rabbit antihemoglobin (1:100; Abcam) at 4 °C overnight. After 3 consecutive washes in PBS (10 minutes each time), the sections were incubated with appropriate fluorescent-conjugated secondary antibodies (1:200; Jackson ImmunoResearch) for 2 hours at room temperature. The number of CD68+Iba1+ or CD206+Iba1+ positive cells in 3 different fields of the right basal cortex was identified and counted from 5 random coronal sections per rat, and positive cells were quantified under microscope fields at ×400 magnification.
8
Immunofluorescence Staining of Microglia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For primary microglia, after indicated treatment, cells were fixed with 4% paraformaldehyde for 30 min and then permeabilized by 0.5% Triton X-100 for 30 min. Following blocking with 5% BSA for 1 h, cells were incubated with indicated primary antibodies overnight at 4°C. After three washes with PBS, cells were stained with DayLight 488/CY3-conjugated secondary antibodies (1:400, Abbkine) for 1 h. DAPI was used to stain nuclei. Images were recorded by Leica confocal microscopy. For brain tissue, double immunofluorescence staining was performed on formalin-fixed, paraffin-embedded brain sections. After blocking with 5% BSA, 4 μm thick paraffin brain slices were incubated with rabbit anti-Caspase-1 (1:200, Abclonal), rabbit anti-GSDMD (1:200, Abclonal), or mouse anti-Iba-1 (1:200, Abcam) antibodies overnight at 4°C. Then, the slices were incubated with CY3-conjugated duck anti-rabbit or DayLight 488-conjugated duck anti-mouse secondary antibodies (1:400, Abbkine) for 1 h at 37 °C. After washing with PBS, DAPI was used to label the nuclei. The number of double-positive cells was detected under a fluorescence microscope.
9
Immunohistochemistry of Recurrent Tumors
10
Multimarker Immunofluorescence Staining
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!