Protocol - Ebp1 immunostaining
Immunohistochemistry for Ebp1 (ABC method)
Category:
In situ hybridization and Immunochemistry
Creation date: Sep 28, 2017
Last revision: May 23, 2018
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
|
Rehydrate slides in Xilene I
|
55-60 °C
|
5'
|
Pre-warm xilene at 55-60°C
|
|
Rehydrate slides in Xilene II
|
55-60 °C
|
5'
|
Pre-warm xilene at 55-60°C - Check that paraffin disappears
|
|
Rehydrate slides in EtOH 100% I
|
RT
|
5'
|
Sections become white
|
|
Rehydrate slides in EtOH 100% II
|
RT
|
5'
|
|
|
Rehydrate slides in EtOH 95%
|
RT
|
5'
|
|
|
Rehydrate slides in EtOH 75%
|
RT
|
5'
|
|
|
Rehydrate slides in EtOH 50%
|
RT
|
5'
|
|
|
Rinse the slides in tap water
|
RT
|
||
|
Boiling step 1 (in 1x citrate buffer)
|
700 watt
|
4'
|
in microwave
|
|
Boiling step 2 (in 1x citrate buffer)
|
700 watt
|
1' 30"
|
in microwave
|
|
Boiling step 3 (in 1x citrate buffer)
|
700 watt
|
1' 30"
|
in microwave
|
|
Boiling step 4 (in 1x citrate buffer)
|
700 watt
|
1' 30"
|
in microwave
|
|
Cooling down
|
RT
|
let the slides cool down adding tap water gradually
|
|
|
Wash in H2O
|
RT
|
5'
|
|
|
Peroxidase blocking
|
RT
|
30'
|
|
|
Wash in 1x PBS
|
RT
|
5'
|
|
|
Incubation with blocking solution
|
RT (in humid chamber)
|
1h
|
One by one, dry a bit each slide, surround the sections with a DAKO pen and put the slides in a humid chamber. DO NOT let the the sections to dry. Consider a V of 200ul for each slide.
|
|
Incubation with primary antibody
|
4°C (in humid chamber)
|
O.N.
|
rabbit-anti Ebp1 antibody (Abcam polyclonal 1:2500)
|
|
Wash in 0.1% Tween - 1xPBS
|
RT
|
5'
|
|
|
Incubation with secondary antibody
|
RT (in humid chamber)
|
1h
|
DAKO biotynilated anti-rabbit (1:400)
|
|
Wash in 0.1% Tween - 1xPBS
|
RT
|
5'
|
|
|
Wash in 0.1% Tween - 1xPBS
|
RT
|
5'
|
|
|
Incubation with AB complex
|
RT
|
30'
|
Vectastain ABC kit - Prepare the mix 30' before incubation
|
|
Wash in 0.1% Tween - 1xPBS
|
RT
|
5'
|
|
|
Wash in 0.1% Tween - 1xPBS
|
RT
|
5'
|
|
|
Reveal the signal
|
RT
|
max 10' (usually 2')
|
using DAB solution (Vektor DAB kit) - control the reaction under the microscope
|
|
Stop the reaction in water
|
RT
|
||
|
Counterastain with haematoxilyn
|
RT
|
1'
|
|
|
Put the slides under flowing water
|
RT
|
from 6' to 10'
|
|
|
Dehydrate slides in EtOH 50%
|
RT
|
5'
|
|
|
Dehydrate the slides in EtOH 75%
|
RT
|
5'
|
|
|
Dehydrate the slides in EtOH 95%
|
RT
|
5'
|
|
|
Dehydrate the slides in EtOH 100%
|
RT
|
5'
|
|
|
Dehydrate the slides in Xilene
|
RT
|
5'
|
|
|
Mount the slides using DEPEX
|
perform this step under a hood
|
Other informations
Boiling proceudure:
Prepare 500 ml of 1X Citrate Buffer/each round of boiling
Prepare the slides in the centre of the rack, fill the empty positions with blank slides
Fill the box with 1X Citrate Buffer up to the top
Put the box in the centre in the microwave and put the lid of the box upside down and a little bit rotated as well as a little bit of 1X Citrate
Buffer in a beaker.
Citrate buffer is a 0.1 M solution of Trisodium Citate pH 6 (Prepare 10X as stock solution)
Blocking Solution
10% Normal Goat Serum - 1% Bovine Serum Albumin in 1X PBS
*This protocol is for PARAFFIN SECTIONS
Quality validation:
Yes
Validation info
The protocol has been used to produce data published in peer-reviewed journals.
Solutions
-
Block endogenous peroxidase (200ml)
Reagents F-concentration Quantity I-concentration H2O20.3%20ml30% (100x)MetOHabs180ml
