Aug 16, 2023
Immunocytochemistry (ICC) for Progenitor markers FOXA2 and OTX2
- Ning Xia1,2
- 1Massachusetts General Hospital;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
Protocol Citation: Ning Xia 2023. Immunocytochemistry (ICC) for Progenitor markers FOXA2 and OTX2. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7ppp3gwz/v1
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 16, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86555
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000312
Abstract
This is the protocol for Immunocytochemistry (ICC) for Progenitor markers FOXA2 and OTX2.
Attachments
Materials
| A | B | C | |
| Blocking Buffer (BP) | 1x PBS 5% BSA 0.3% Triton X-100 | To make 10mL of Blocking Buffer: 0.5g of BSA 30µl of Triton X-100 Fill to 10mL with 1x PBS | |
| 1º antibody (-20ºC fridge) | FOXA2 Rb OTX2 Mus IgG | ||
| 2º antibodies (4ºC fridge) | A11008 Alexa Flour 488 goat anti-rabbit IgG (HHH+L) A10036 Alexa Flour546 donkey anti-mouse IgG (H+L) | Thermo Fisher Cat # A11008 Thermo Fisher Cat # A10036 | |
| DAPI (-20ºC fridge) | Stock 1mg/mL | Final (1µg/mL) | |
| - Each well of both 8µm Chamber slide and 48-well plate should use 200µL of solution | |||
Grow the cells on either 8µm chamber slide or 48-well plate. (1x105cells/well)
- Coating and plating densities can be found in the differentiation kit
Aspirate medium and rinse with 1x PBS
Fixation: Cover cells with warm 4% PFA and fix for 20 min at RT.
- Cover the vessel and place in the hood
Rinse 3X in 1x PBS for 5 min each at the lab bench
Blocking and permeabilize for 30 min at RT with Blocking Buffer
1º antibody incubation dilute in 1/3 blocking buffer + 1x PBS and incubate overnight in the cold room
-20º (box labelled antibodies for iPSCs)
- FOXA2 Rb à (1:250)
- OTX2 Mus IgG à (1:250)
o Add 4µl of antibody per 1mL of prepared antibody solution
Rinse three times in 1x PBS for 5 min each
2º antibody incubation for 1hr at RT in the dark
- A11008 Alexa Flour 488 goat anti-rabbit IgG (HHH+L) 1:400 in PBS
- A10036 Alexa Flour546 donkey anti-mouse IgG (H+L) 1:400 in PBS
o Add 2.5µL of antibody per 1mL of prepared antibody solution
*Combine these two antibodies in one tube. Ex. If you were making 1.6mL (for 8 wells) you would add 4µL of each antibody
Rinse 3X in 1X PBS for 5 min each
Incubate with DAPI at (1µg/mL) for 10min at RT (Stock is 1mg/mL which is 1000x)
a. Add 1µl of Stock DAPI to 1mL of prepared solution.
11. Rinse 2X in 1X PBS and image.