Apr 29, 2024
Immunofluorescence Staining in Mouse Brain Tissue Sections
- madalynn.erb Erb1
- 1Van Andel Research Institute
- Team Lee
Protocol Citation: madalynn.erb Erb 2024. Immunofluorescence Staining in Mouse Brain Tissue Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6zopl5d/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: April 08, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 98951
Keywords: ASAPCRN, immunofluorescence staining in mouse brain tissue section, immunofluorescence staining, mouse brain tissue section, immunofluorescence, staining, tissue
Funders Acknowledgements:
aligning science across parkinsons
Grant ID: 000592
Abstract
This protocol details the immunofluorescence staining in mouse brain tissue sections.
Materials
Pyrex® Staining DishTed Pella Inc.Catalog #36754-60
8-Section Staining NetsTed Pella Inc.Catalog #36154-64
Fisherbrand™ Superfrost™ Plus Microscope SlidesFisher ScientificCatalog #12-550-15
ProLong™ Diamond Antifade MountantInvitrogen - Thermo FisherCatalog #P36961
Primary Antibodies:
| A | B | C | D | |
| Target | Species | Conc | Manufacturer | |
| TH | Rabbit | 1:2000 | Novus Biological N300109 | |
| TH | Chicken | 1:750 | Abcam ab76442 | |
| GFP | Chicken | 1:2000 | Aves Labs- GFP-1010 | |
| GFP | Rabbit | 1:500 | Thermofisher A-11122 | |
| GFP | Mouse | 1:1000 | Roche 11814460001 | |
| P62 | Guinea pig | 1:2000 | Progen GP62-C | |
| LAMP2 | Rat | 1:1000 | Abcam ab13524 | |
| GAD67 | Mouse | 1:500 | Millipore Sigma MAB5406 | |
| parvalbumin | Rabbit | 1:500 | Abcam ab11427 | |
| TFE3 | Rabbit | 1:500 | Abcam ab93808 |
Secondary Antibodies (From Thermofisher):
| A | B | C | D | |
| Antibody | Fluorophore | Concentration | Cat Number | |
| goat-anti rabbit | 488 | 1:500 | A-11008 | |
| goat-anti rabbit | 546 | 1:500 | A-11010 | |
| goat-anti rabbit | 647 | 1:500 | A-21245 | |
| goat-anti mouse | 488 | 1:500 | A-11029 | |
| goat-anti mouse | 546 | 1:500 | A-11003 | |
| goat-anti rat | 647 | 1:500 | A-21247 | |
| goat-anti chicken | 488 | 1:500 | A-11039 | |
| goat-anti chicken | 647 | 1:500 | A-21449 |
Troubleshooting
Day 1
30m
Staining of 35μm free-floating mouse brain sections is performed in glass staining dishes (Pyrex 36754-60) using 8-section staining nets (Ted Pella 36154-64). dx.doi.org/10.17504/protocols.io.5jyl8pzk9g2w/v1
The sections can be transferred between wells using a paint brush.
Volume of solution:
- 20 mL -30 mL for antibodies.
- 50 mL for washing.
Wash sections 3 times (5 min each wash) in Phosphate Buffer Saline (PBS) at Room temperature to remove cryo-protectant solution.
Wash sections for 00:05:00 in Phosphate Buffer Saline (PBS) at Room temperature to remove cryo-protectant solution (1/3).
5m
Wash sections for 00:05:00 in Phosphate Buffer Saline (PBS) at Room temperature to remove cryo-protectant solution (2/3).
5m
Wash sections for 00:05:00 in Phosphate Buffer Saline (PBS) at Room temperature to remove cryo-protectant solution (3/3).
5m
Wash sections 3 times (5 min each wash) in PBS + 0.1% triton X-100.
Wash sections for 00:05:00 in PBS + 0.1% triton X-100 (1/3).
5m
Wash sections for 00:05:00 in PBS + 0.1% triton X-100 (2/3).
5m
Wash sections for 00:05:00 in PBS + 0.1% triton X-100 (3/3).
5m
Block sections for 01:00:00 in PBS + 10% Normal Goat Serum (NGS) + 0.4% Bovine Serum Albumin (BSA) and 0.2% triton X-100 for 01:00:00 at Room temperature .
2h
Incubate sections with primary antibodies in blocking solution (PBS + 0.1% triton X-100, 0.4% BSA) Overnight at 4 °C .
1h
Day 2
2d 2h 5m
Wash sections 3 times (10 min each wash) in PBS + 0.1% triton X-100 at Room temperature .
Wash sections for 00:10:00 in PBS + 0.1% triton X-100 at Room temperature (1/3).
10m
Wash sections for 00:10:00 in PBS + 0.1% triton X-100 at Room temperature (2/3).
10m
Wash sections for 00:10:00 in PBS + 0.1% triton X-100 at Room temperature (3/3).
10m
Incubate with fluorescent secondary antibodies (1:500) diluted in PBS + 0.1% triton X-100 + 0.4% BSA for 2 hours at Room temperature or 24:00:00 at 4 °C .
Note
Sections should be protected from light at this step and all proceeding steps.
1d
Wash sections 3 times (10 min each wash) in PBS + 0.1% triton X-100 at Room temperature .
Wash sections for 00:10:00 in PBS + 0.1% triton X-100 at Room temperature (1/3).
10m
Wash sections for 00:10:00 in PBS + 0.1% triton X-100 at Room temperature (2/3).
10m
Wash sections for 00:10:00 in PBS + 0.1% triton X-100 at Room temperature (3/3).
10m
Incubate with DAPI in PBS (1:5000) for 00:30:00 at Room temperature .
30m
Wash sections 3 times (10 min each wash) in PBS at Room temperature .
Wash sections for 00:10:00 in PBS at Room temperature (1/3).
10m
Wash sections for 00:10:00 in PBS at Room temperature (2/3).
10m
Wash sections for 00:10:00 in PBS at Room temperature (3/3).
10m
Mount sections:
Use a petri dish filled with PBS to mount the sections on superfrost plus slides (Fisher Scientific 12-550-15).
This is easiest to do using a medium sized paint brush.
Let sections dry 00:02:00 -00:05:00 .
Note
Coverslip using ProLongTM Diamond Antifade Mountant (Thermofisher P36961).
7m
Dry slides for 24:00:00 at Room temperature .
- Protect slides from light at this step.
- Store slides at 4 °C .
1d