Brains were fixed overnight in 4% paraformaldehyde (PFA) at 4°C, followed by submersion in 30% sucrose until sinking, as previously described (Silver et al. 2010 (link)). Brain cryostat sections (20 µm) were prepared and stored at −80°C until use. Sections were permeabilized with 0.25% Triton X-100 for 10 min and blocked with MOM block reagent (Vector laboratories) for 1 h at room temperature (RT). Sections were incubated with primary antibodies for 2 h at RT or overnight at 4°C. Sections were then incubated in species appropriate secondary antibodies and Hoechst for 15 min at room temperature. The following primary antibodies were used: rabbit anti-CC3 (diluted 1:200; Cell Signaling); rabbit anti-TBR2 (1:1,000; Abcam); rabbit anti-PAX6 (1:1,000; Millipore); mouse anti-TUJ1 (1:400; Covance). The following secondary antibodies were used: Alex Fluor 488 and Alex Fluor 594 (1:400; Invitrogen). Hoechst (Thermo Fisher Scientific) was used for nucleus counterstain. High-magnification images were captured using a Zeiss Axio Observer Z.1 microscope coupled with an apotome. Cortical thickness was measured with Zen software. Cell quantification was performed with ImageJ/FIJI. Three sections from anatomically comparable regions per embryo and three biological replicates from control (wild-type) and mutant alleles (Casc3RRU345/+, Casc3RRU345/RRU345, and Casc3RRU345/Null) were quantified.
Rabbit anti cc3
Manufactured by Cell Signaling Technology
Rabbit anti-CC3 is a primary antibody that specifically detects cleaved caspase-3, a key executioner caspase involved in apoptosis. It recognizes the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage at Asp175.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst, by Thermo Fisher Scientific (1 mentions)
Alex fluor 488, by Thermo Fisher Scientific (1 mentions)
Rabbit anti pax6, by Merck Group (1 mentions)
Alex fluor 594, by Thermo Fisher Scientific (1 mentions)
Mom block reagent, by Vector Laboratories (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Mouse anti tuj1, by Fortrea (1 mentions)
Rabbit anti tbr2, by Abcam (1 mentions)
Rm2125rt, by Leica (1 mentions)
Nile red, by Merck Group (1 mentions)
Alexa 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gm130, by Abcam (1 mentions)
Chicken anti gfp, by Abcam (1 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti ctbp2, by BD (1 mentions)
Vibrotome, by Leica (1 mentions)
Alexa488 conjugated mouse anti tuj1, by Fortrea (1 mentions)
Rabbit anti 53bp1, by Novus Biologicals (1 mentions)
Anti g3bp1, by Proteintech (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
6 protocols using rabbit anti cc3
1
Tissue Fixation and Immunohistochemistry of Mouse Brain
2
Quantifying Neuronal Apoptosis in Fetal Brains
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Neuronal apoptosis was analyzed as previously described (Abramowski et al., 2018 (link)). E15.5 fetal heads were fixed overnight at 4°C by immersion in 4% paraformaldehyde and embedded in paraffin with a Tissu-tek processor (VIP, Leica). 5 μm coronal sections were then obtained using a microtome (Leica RM2125RT) and mounted onto glass slides for histological analyses. After paraffin removal and citrate treatment, the brain sections were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 15 min and incubated for 2 hr with 7.5% fetal bovine serum and 7.5% goat serum in PBS. The sections were incubated with rabbit anti-CC3 (Cell Signaling 9661) overnight at 4°C. After washing, the sections were incubated with goat anti-rabbit Alexa Fluor 488 or 594 conjugated secondary antibody (ThermoFisher) for 1 hr. After washing, nuclear staining was achieved by incubation with 4′-6-diamidino-2-phenylindole (DAPI) to quantify apoptosis induction by the detection of pyknotic nuclei (Roque et al., 2012 (link)). Slides were mounted under Fluoromount (Southern Biotechnologies Associates). Tissues were examined under a fluorescence microscope (50i, Nikon, Japan) with a 10× (NA = 0.3) objective in three channels (appearing red, green, and gray) as separate files. These images were then stacked with Photoshop software (Adobe).
3
Immunofluorescence Staining of Malaria Parasites
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The dyes and primary antisera employed in this study were as follows: DAPI (4′,6-diamidino-2-phenylindole; Life Technologies) (1:50), Rh-WGA (Vector Labs) (1:40), Nile red (Sigma) (1:50), phalloidin-Alexa 488 (Life Technologies) (1:10), chicken anti-GFP (Abcam ab13990), mouse anti-CSP (gift from Photini Sinnis), rabbit anti-mtTFA (Santa Cruz H-203), rabbit anti-TRAP (gift from Photini Sinnis), rabbit anti-CC3 (catalog no. 9661; Cell Signaling), AAPP (gift from Hiroyuki Matsuoka), and rabbit anti-GM130 (Abcam ab30637). Secondary antibodies (goat anti-rabbit 488 [A11008] and goat anti-mouse 647 [A21235]) were from Life Technologies.
4
Immunofluorescence Staining of Inner Ear
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For whole-mount beta-III Tubulin (Tuj-1), Myosin6 (Myo6), C-terminal binding protein 2 (CtBP2), Cleaved Caspase-3 (CC3), and Parvalbumin (PV) staining, mouse heads at desired time points were fixed with 4% PFA for 2–24 hours. Fixed cochleae were dissected out and permeabilized with 0.5% TrionX-100 followed by incubation in 10% serum blocking buffer for at least 1 hour at room temperature. Incubation of primary antibody was carried out for overnight at 4 °C, followed by Alexa-conjugated secondary antibodies (1:1000, Invitrogen). For cross-section staining, mouse heads were fixed with 4% PFA for overnight. Fixed inner ear bone structure was dissected out and cryoprotected with sucrose. Tissue was then embedded in OCT (Tissue-Tek) and cut into 35-μm sections using a vibrotome (Leica). Sections were later stained using Tuj-1, Myo6, and PV as the whole-mount staining procedure. Antibodies used in this study and their dilution were as followed: Alexa488-conjugated mouse anti-Tuj1 (1:300; Covance), rabbit anti-PV (1:300; Swant Inc.), rabbit anti-Myo6 (1:500, Millipore), mouse anti-CtBP2 (1:250, BD Biosciences), rabbit anti-CC3 (1:300, Cell Signaling Technology).
5
Neuronal Markers Immunostaining Protocol
6
Pericyte Apoptosis in Neuroinflammation
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Primary mouse brain vascular pericytes were obtained from iXCells Biotechnologies (10MU-014). All experiments were performed within five passages, wherein cells were grown on collagen-coated (Sigma) chambered slides in pericyte medium containing 2.5% FBS, penicillin-streptomycin, and pericyte growth solution (ScienCell). Once the cells reached ∼70% confluence, they were treated with either DMSO (vehicle) or 100 ng/ml IFN-γ (R&D) for 48 h. Immunocytochemistry was performed by fixing cultures with 4% PFA for 10 min, followed by incubation with the primary antibodies rabbit anti-CC3 (1:100; Cell Signaling), rabbit anti-Pdgfrβ (1:100; Cell Signaling), and rabbit anti-pSTAT1 (1:100; Cell Signaling) for 1 h at room temperature. The samples were incubated with appropriate Alexa Fluor secondary antibody and DAPI and then imaged using a Zeiss 780 LSM confocal microscope. The number of apoptotic pericytes was determined by counting the number of CC3+ cells versus the total number of DAPI+ cells using Zen software (a minimum of three 20× images were captured and analyzed per experiment; three independent experiments were performed).
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