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Home  |  Protocols  |  In situ hybridization and Immunochemistry  |  Ebp1 immunostaining

Protocol - Ebp1 immunostaining

Immunohistochemistry for Ebp1 (ABC method)

CategoryIn situ hybridization and Immunochemistry
Creation dateSep 28, 2017
Last revisionMay 23, 2018
Contact
NameMaria Donnini
Address111, via Pietro Castellino
Emailmaria.donnini@igb.cnr.it

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
    H2O2
    0.3%
    20ml
    30% (100x)
    MetOHabs
    180ml

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