Aug 08, 2023
Immunohistochemistry for p-eIF2a and eIF2a, v.230804
This protocol is a draft, published without a DOI.
- elanalockshin1
- 1Calakos Lab, Duke University
Protocol Citation: elanalockshin 2023. Immunohistochemistry for p-eIF2a and eIF2a, v.230804. protocols.io https://protocols.io/view/immunohistochemistry-for-p-eif2a-and-eif2a-v-23080-cycsxswe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 08, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86130
Keywords: ASAPCRN
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Abstract
Authors:
Callie Eatman, Elana Lockshin with recommendations from Xinlu Ding, Dan lab, UC-Berkeley
Calakos lab
Duke University
Reagents
Reagents
phospho-eIF2a antibody: Abcam ab32157
eIF2a antibody: Cell signaling #5324
Tissue Prep
Tissue Prep
Brains are stored in 4% PFA for 24hrs in 4C before storage in 1xPBS in 4C until sectioning
Brain is sectioned in 1xPBS bath into 40um slices using Vibratome.
Slices are stored in 0.03% Sodium Azide in 1xPBS in 24 well plates in 4C.
IHC
IHC
In 24 well plate/ 2 slices per well with a minimum of 300uL of solution in well
Day 1:
1. Alkaline Retrieval: 20 min in 1% NaOH (in 1x PBS) on shaker (RT) (make fresh each time, do not store in glass)
2. 3x10 min wash on shaker in PBS-T (PBS with 0.3% Triton X-100) (RT)
3. 2 hr blocking in 10% NDS/1% BSA in PBS-T (RT)
4. Primary antibody in dilution buffer (5% NDS +1%BSA) in PBS-T 48hrs in 4C on shaker
a. P-eIF2a Antibody (dilution 1:100 )
OR
b. eIF2a Antibody (dilution: 1:500 )
Day 2:
Day 2:
1. 3x10 min wash on shaker in PBS-T (RT)
2. Secondaries for 2hrs in dilution buffer (5% NDS +1%BSA) in PBS-T (RT)
● DK~Rb Alexa fluor 488+ (1:500)
3. 3x10 min wash on shaker in PBS-T (RT)
○ (for Hoescht: 1:750 in 1st 10 min wash)
4. Mount on slides by floating section in PBS and let sit until dry
5. Coverslip with Vectashield hardset mounting medium
6. Seal slide with nail polish next day
7. Store in 4C