The largest database of trusted experimental protocols

2 protocols using mouse monoclonal anti β 3 tubulin tuj1

1

Immunofluorescence Staining of Primary Neurons

Check if the same lab product or an alternative is used in the 5 most similar protocols
For all IF stainings, primary neurons on coverslips were fixed for 15 min in 4% paraformaldehyde (PFA) at room temperature. Cells were permeabilised and blocked in PBS containing 10% donkey serum (SouthernBiotech, 0030-01) and 0,1% Triton X-100 for (108603 Merck Millipore) for 1 h at room temperature during gentle shaking. Incubation with primary antibodies in PBS-Triton 0.1% with 5% donkey serum was performed overnight at 4 °C in the following dilutions: mouse monoclonal anti-β-III tubulin (Tuj1) (1:1000; BioLegend # 801202), rabbit anti-cleaved caspase-3 (1:1000; Cell Signaling Technology # 9661), and mouse anti-Cdk7 (1:100, Santa Cruz # sc-7344). After three washing steps with PBS, secondary antibodies (Alexa FluorTM, Invitrogen) were used 1:1000 in PBS with 5% donkey serum and 0.1% Triton X-100 for 1 h at room temperature. Cells were washed two more times with PBS, counterstained for 10 min with DAPI 1:5000 (D9542 Sigma), and mounted with fluorescence mounting medium (Dako, S3023) before confocal microscopy (Olympus FV1000).
+ Open protocol
+ Expand
2

Immunostaining of Neural Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols
The cultures were fixed in 4% paraformaldehyde in PBS for 1 hr at room temperature (RT) and incubated in icecold 20% methanol in PBS for 5 min at RT. Fixed specimens were permeabilized in 0.5% Triton X-100 in PBS overnight at 4 C, and block in 15% basal serum albumin (BSA; Sigma) in PBS for overnight at 4 C. Specimens were incubated in primary antibodies with 1:500 dilution for 72 hr at 4 C as follows: mouse monoclonal anti-Nestin (Biolegend), mouse monoclonal anti-βIII-tubulin (Tuj1; Biolegend), goat polyclonal anti-synaptic vesicle glycoprotein 2A (SV2A; Santa Cruz), rabbit polyclonal anti-tropomyosin receptor kinase B (TrkB; Santa Cruz), and mouse monoclonal anti-GATA binding protein 3 (Gata3; Biolegend). After 3 times of 5 min washing with PBS, appropriate secondary antibodies were used to conjugate the primary antibodies for 4 hr at RT with 1:1,000 dilution, including goat anti-mouse Alexa Fluor 488, goat anti-mouse Alexa Fluor 594, donkey anti-goat Alexa Fluor 594, and donkey anti-rabbit Alexa Fluor 594 (all are from Molecular Probes). All antibodies were diluted in 5% BSA and 0.05% Tween-20 in PBS. All specimens were mounted in ProLong Diamond Anti-fade Mounting with DAPI (Molecular Probes) prior to observing by BX53 Upright Microscope (Olympus) and imaged by cellSens software (Olympus).
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!