Mice were perfused with 4 % PFA, brains were removed and immersion fixed. Brain sections were cut using a vibratome (100 μm) and incubated in 0.5 % gelatin, 5 % horse serum and 0.01 % saponin in PBS for 5 h. Microglia staining was performed using a Rb anti-Iba1 (1:600; Wako), a secondary biotinylated anti-Rb (1:500; Rockland), streptavidin-HRP (1:1000; Rockland, S000-03) for 3 h and visualized using DAB Substrate Kit (Cell Marque, 957D). Tissue was then fixed with 2 % PFA, 2.5 % GA and sections were processed using routine EM protocols. Grids were not counterstained to preserve immunostaining. Finished blocks were sectioned using a DiATOME ultra 45° diamond knife and a LEICA Ultracut UC7. Sections (90 nm) were cut and picked up onto 200 hex mesh, formvar-carbon coated copper grids (Ted Pella, 01800-F). Images were captured using a JEOL 1200 EX II Transmission Electron Microscope with AMT digital camera.
Rb anti iba1
Manufactured by Fujifilm
The Rb anti-Iba1 is a laboratory reagent used for the detection and localization of Iba1, a protein involved in the regulation of macrophage and microglial activity. It is a rabbit polyclonal antibody that can be utilized in various immunohistochemical and immunocytochemical applications.
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Lab products found in correlation
880 confocal microscope, by Zeiss (3 mentions)
Dapi counterstain, by Southern Biotech (3 mentions)
Fish gelatin, by Merck Group (2 mentions)
Ultracut uc7, by Leica (1 mentions)
Dab substrate kit, by Cell Marque (1 mentions)
Ex 2 transmission electron microscope, by JEOL (1 mentions)
Streptavidin hrp, by Rockland Immunochemicals (1 mentions)
Alexa fluor 647 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 donkey anti goat, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
5 protocols using rb anti iba1
1
Ultrastructural Analysis of Microglia
2
Immunostaining of Murine Brain Sections
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Mice were anesthetized using isoflurane overdose and then perfused with 20 mL ice-cold 1× PBS, followed by 50 mL 4%PFA. Brains were postfixed overnight in PFA and then cryopreserved and sectioned as previously described (14 (link)). The 30 μm serial sections were blocked in 2% cold water fish gelatin (MilliporeSigma.) in 0.2% Triton for 1 hour and then incubated with Gt Anti-Tie2 (R&D Systems) Ab (1:200), Rb anti-Iba1 (Wako) Ab (1:200), Rb anti-Ccr2 (Abcam) Ab (1:200), and/or Gt anti-arginase-1 (1:500) in block overnight; washed with 1× PBS; and then treated with appropriate secondary Abs (Invitrogen): Alexa Fluor donkey anti-rabbit 594, Alexa Fluor donkey anti-rabbit 647, Alexa Fluor donkey anti-goat 594, and Alexa Fluor donkey anti-goat 647 (1:250 in block) for 1 hour. The sections were washed and mounted in media with DAPI counterstain (SouthernBiotech). Images were acquired using a Zeiss 880 confocal microscope (Carl-Zeiss).
3
Immunohistochemical Analysis of Microglia and Associated Markers
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Serial coronal sections were washed in PBS, subsequently blocked 2% cold water fish gelatin/0.2% Triton X-100 (Sigma, Inc.) for 2 hours, and then incubated overnight with the following primary antibodies diluted in blocking solutions: Rb anti-IBA1 (Wako) antibody (1:250), Rt anti-IBA1 (Abcam) antibody (1:250), Rt anti-Mertk (ThermoFisher) antibody (1:200), Rb anti-Mertk (Abcam) antibody (1:200), Rb anti-NeuN (cell signaling) antibody (1:200), Rb anti-P-ERK (cell signaling) antibody (1:200), or Rb anti-P-Stat6 (cell signaling) antibody (1:150). Afterward, the sections were washed with 1× PBS and incubated with the appropriate secondary antibodies (Invitrogen) diluted (1:250) in the blocking solution for 1 hour: AlexFluor donkey anti-rabbit-594, AlexFluor donkey anti-rat-647, AlexFluor donkey anti-rat-594, or AlexFluor donkey anti-rabbit-647. Following another round of washing with 1× PBS, the sections were mounted using a media containing DAPI counterstain (SouthernBiotech). Confocal images were acquired using a Zeiss 880 confocal microscope (Carl Zeiss, Oberkochen, Germany).
4
TUNEL Staining of Brain Sections
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TUNEL staining of serial coronal sections of perfused fixed brains was performed using the Click-iT Plus TUNEL Assay; 647 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Serial coronal sections (300 μm) were permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase enzyme followed by Alexa Fluor 647-conjugated nucleotides. Following the TUNEL reaction, the sections were blocked in 2% cold water fish gelatin (Sigma, Inc.) in 0.2% triton for 1 h, then incubated with Rb anti-IBA1 (Wako) or Rb anti-NeuN (cell signaling) antibody (1:200 in blocking solution) overnight. Slides were then washed with 1×PBS, treated with AlexFluor donkey anti-rabbit-555 (1:250 in block) for 1 hour, further washed in 1×PBS, and then mounted in media with DAPI counterstain (SouthernBiotech). Z-stack images were acquired using a Zeiss 880 confocal microscope (Carl-Zeiss, Oberkochen, Germany).
5
Iba1 Immunostaining of Brain Sections
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Animals were transcardially perfused with saline 0.9% under isoflurane anesthesia. After perfusion, brains were fixed for 24 h in 4% paraformaldehyde (PFA), whereafter they were stored in 1% PFA, until further use. Brains were cryoprotected by overnight incubation in 20% sucrose solution and cryosections of 50 lm thick were made. Free floating sections were blocked for 1 h with 5% normal goat serum and thereafter incubated with primary antibody against Iba1 (Rb-anti-Iba1, Wako, Cat.#019-19741) for 72 h at 4 °C. Detection of primary antibody was done by incubation with Alexa fluor 488 donkey anti rabbit (Invitrogen, Cat.#A21206) secondary antibody. After secondary antibody incubation, sections were washes, incubated in Hoechst solution for 5 min, washed again and mounted on glass slides.
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