In situ hybridization was performed by RNAscope™ Multiplex Fluorescent V2 Assay (ACDBio) technology in combination with the RNA-Protein Co-Detection kit (ACDBio), according to the manufacturer’s instructions. In brief, 4 μm paraffin embedded sections were dehydrated, blocked for 10 minutes at room teperature with H2O2, and processed with 1X Antigen Retrieval co-detection buffer for 15 minutes at 99°C. Sections were incubated with Ecad (1:250, #610182, BD Biosciences) overnight at 4°C. Hybridization was performed with a probe targeting mouse Clu (Cat#427891, ADCBio), and the probe signal was developed with Vivid dye 520 nm (1:1500). Sections were counterstained by DAPI and anti-mouse Alexa 633 (1:250, Invitrogen) and mounted with ProLong™ Gold Mountant (ThermoFisher). Samples were imaged on the Stellaris 5 Confocal Microscope (Leica) and images were processed with ImageJ.
Anti mouse alexa 633
Manufactured by Thermo Fisher Scientific
The Anti-mouse Alexa 633 is a fluorescent dye conjugated to an antibody that specifically binds to mouse proteins. It is designed for use in various immunofluorescence techniques, such as flow cytometry and fluorescence microscopy, to detect and visualize mouse target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Sp5 confocal microscope, by Leica camera (3 mentions)
A11039, by Thermo Fisher Scientific (2 mentions)
Vectashield medium, by Vector Laboratories (2 mentions)
E cad, by BD (1 mentions)
Stellaris 5 confocal microscope, by Leica camera (1 mentions)
Prolong gold mountant, by Thermo Fisher Scientific (1 mentions)
Ab16059, by Abcam (1 mentions)
Fluoromount aqueous mounting medium, by Merck Group (1 mentions)
A11011, by Thermo Fisher Scientific (1 mentions)
Nb300 575, by Novus Biologicals (1 mentions)
Anti rabbit alexa488, by Thermo Fisher Scientific (1 mentions)
Anti chicken alexa488, by Thermo Fisher Scientific (1 mentions)
A21052, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexa 568, by Thermo Fisher Scientific (1 mentions)
Mouse anti pi 4 p, by Echelon Biosciences (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Ab13970, by Abcam (1 mentions)
Ab171380, by Abcam (1 mentions)
Anti mouse alexa555 secondary antibody, by Thermo Fisher Scientific (1 mentions)
A21422, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti mouse alexa 633
1
RNAscope-based In Situ Hybridization
2
Immunostaining of Proteins and Lipids
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Immunostaining of proteins and lipids was used in fixed cells with 4% (wt/vol) paraformaldehyde. For surface proteins, nonpermeabilized cells were directly blocked with goat serum and then incubated with the appropriate primary antibody. For internal proteins and lipids, cells were permeabilized with 0.1% (vol/vol) Triton X-100 before blocking and incubation with the primary antibody. Control experiments were performed to make sure that chemical fixation did not induce permeabilization (see Fig. S1 B ). For surface GluA1 analysis, isolated primary dendrites close to the soma of the neuron were selected. Rabbit anti-GluA1 (1:30; PC246) was from Calbiochem. Mouse anti-PI(4)P (1:100; Z-P004) was from Echelon. Mouse anti-TGN38 (1:100; NB300-575) was from Novus Biologicals. Rabbit anti-TGN46 (1:100; ab16059) and chicken anti-GFP (1:1,000; ab13970) were from Abcam. Rabbit anti-SAC1 (1:100; 13033–1-AP) was from Proteintech. Incubation with the appropriate Alexa Fluor–conjugated secondary antibodies was also performed. Anti-mouse Alexa633 (1:100; A21052), anti-rabbit Alexa488 (1:300; A11008), anti-chicken Alexa488 (1:1,000; A11039), and anti-mouse Alexa568 (1:150; A11011) were from Invitrogen. Coverslips were mounted with Fluoromount Aqueous Mounting Medium (F4680; Sigma-Aldrich).
3
Immunostaining and Confocal Imaging of Drosophila Brains
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Brains were dissected in ice-cold PBS and fixed for 20 min in 4% paraformaldehyde in PBS, then stained as described 40 (link). Brains were incubated for 1-2 days with primary antibodies against GFP (chicken, abcam 13970, diluted 1:2000) and Bruchpilot (mouse, DSHB nc82, diluted 1:50) followed by 1-2 days with secondary antibodies (anti-chicken Alexa 488 diluted 1:1000 and anti-mouse Alexa 633 diluted 1:200, Life Technologies), interspersed with washes in PBS + 0.3% Triton. Brains were mounted in Vectashield medium (Vector Labs), then the immunostained fluorescent signal and (when present) native RFP fluorescence were imaged using a Leica SP5 confocal microscope at 25X magnification, using manually adjusted laser and gain settings.
4
Immunostaining and Confocal Imaging of Drosophila Brains
5
Whole Mount Immunofluorescence for Neurotransmitter Markers
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Whole mount immunofluorescence was performed as described in Ref.69 (link). Following FISH, a rabbit polyclonal anti-TH primary antibody22 (link) was used at 1:500 dilution and detected with an anti-rabbit Alexa555-conjugated secondary antibody (2 µg/ml, Life Technologies A-21428). For double FIHC (Tg(top:dGFP)w25 and Tg(7xtcf-Xla.siam:gfp)ia4 embryos), a chicken anti-GFP antibody (5 µg/ml; Invitrogen) was combined with a polyclonal rabbit anti-TH antibody22 (link). Primary antibodies were detected with an anti-chicken Alexa488 antibody (2 µg/ml; Life Technologies A11039) and an anti-rabbit Alexa555 antibody (2 µg/ml, Life Technologies A21430). Following FISH for sox2 and sox3, primary mouse-anti-Sox2 antibody (2.5 µg/ml, Abcam, ab171380) was used and subsequently detected using an anti-mouse-Alexa633 (2 µg/ml, Life Technologies A21050) antibody. Following EdU detection, the Sox2 antibody was detected using an anti-mouse-Alexa555 secondary antibody (2 µg/ml, Life Technologies A21422).
6
Immunohistochemistry of Drosophila Brains
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Immunohistochemistry was performed as described previously30 (link). Briefly, 3–7-day old brains were dissected in ice-cold PBS, fixed in 4% paraformaldehyde, and blocked in PBST (PBS and 0.3% Triton X-100) with 5% normal donkey serum (Jackson Laboratory) before staining. Brains were then incubated with 1:1,000 rabbit anti-GFP (Invitrogen) and 1:50 mouse anti-nc82 (Developmental Studies Hybridoma Bank) antibodies overnight at 4 °C and washed five times in PBST. Brains were then incubated with 1:1,000 Alexa 568 anti-rabbit (Life Technologies) and 1:1,000 Alexa 633 anti-mouse (Life Technologies) antibodies for 4 h at room temperature before washing five times in PBST and coverslip mounting in Vectashield (Vector Labs). Images were taken at × 40 magnification on a Leica SP5 confocal microscope at 0.5 mm intervals and reassembled for display as maximum projections using Fiji56 (link).
7
3D Co-culture and Tight Junction Staining
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Human macrophages or dendritic cells were pre-stained with 2 mg/ml of 6-carboxyfluorescein diacetate before they were used in the co-culture. The 3D co-culture was fixed with 3% paraformaldehyde. Tight junctions were stained with monoclonal mouse anti-occludin (Zymed, San Francisco, CA; 1:200) and Alexa633 anti-mouse (Life Technologies, Darmstadt, Germany; 1:500), nuclei were stained with DAPI (Life Technologies, Darmstadt, Germany). Detailed protocol is described in SI.
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