Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue (Brain)
Permeabilization
No
Specification
Brain
Blocking step
Blocking Buffer as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde
Other product details
Incubation time
12 hour(s) and 0 minute(s) · Temperature: 4°C
Dilution
1/500
Secondary antibody
Dilution
1/100
Name
Non-Abcam antibody was used: anti-Rabbit-HRP
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 568
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 568
Additional data
Additional Notes
Experiment procedure:
1. Rinse tissue with PBS for 5 mins at room temperature (RT)
2. Incubate tissue with 0.5% triton 100 PBS solution for 60 mins
3. Incubate tissue with 1% hydrogen peroxide PBS solution for 60 mins at RT
4. Incubate tissue with 1% blocking buffer* for 2 hrs at RT
5. Incubate tissue with 5HT3A antibody solved in PBS with 0.5 M NaCl at 4 °C overnight at dilution 1:500
6. Rinse tissue in PBS with 0.5 M NaCl and 0.5% Tween 20 for 5 mins three times and PBS once
7. Incubate with anti-Rabbit-HRP* at 1:100 dilution in 1% blocking buffer for one hour
8. Rinse tissue with PBS for 5 mins three times
9. Incubate tissue with TSA plus Cyanine 5** reagent for 15 mins
10. Rinse tissue with PBS for 5 mins three times
11. Mounting tissue on glass slide with mounting medium with DAPI***
* Commercial available at Life Technology, Cat. No. T-20914
** Commercial available at Perkin Elmer, Cat. No. NEL745001KT
*** Commercial available at Vector Laboratory, Cat. No. H-1200
Comments: It is possible to decrease the background by incubating 5HT3A antibody with tissue at high salt concentration environment and rinse tissue with stringent condition (PBS with high salt concentration and detergent). Combining with commercial available Tyramide Signal Amplification kits as indicated, we were able to get clear signal as attached.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
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投稿 Sep 25 2014