Jul 31, 2024
Immunohistochemistry against p62 and LBP110
- 1Leibniz Institute on Aging – Fritz Lipmann Institute (FLI)
Protocol Citation: Dennis de Bakker 2024. Immunohistochemistry against p62 and LBP110. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32b47v25/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: July 30, 2024
Last Modified: July 31, 2024
Protocol Integer ID: 104292
Keywords: Immunohistochemistry, mouse hippocampus, cryosections
Abstract
Immunohistochemistry staining co-labeling LBP110 and p62/SQSTM1 and DAPI.
Materials
Buffer PEM (pH 7.4):
80 mM PIPES (Roth 9156.3)
5 mM EGTA (AppliChem (A0878,0025))
1 mM MgCl2 (Merck A748033 703)
Buffer PEMTx:
PEM (described above) + 0.2% Triton X-100
Sodium citrate solution (10mM) (pH 6.0)
10mM sodium citrate
Adjust pH to 6 with 1M Citric Acid
Blocking buffer (to make 1mL, which is good for 2 slides):
890ul PEMTx
100ul Fetal Calf Serum
10ul DMSO
Antibodies:
- Mouse anti-p62/SQSTM1 (clone 2C11) (Novus Biologicals, catalog no. H00008878-M01). Used 1:500.
- Rabbit anti-LBP110. Used 1:100. Antibody produced as described in: Kibbey, M. C., Jucker, M., Weeks, B. S., Neve, R. L., van Nostrandi, W. E., & Kleinman, H. K. (1993). f8-Amyloid precursor protein binds to the neurite-promoting IKVAV site of laminin. In Proc. Natl. Acad. Sci. USA (Vol. 90).
- Anti-mouse Alexa555 (used 1:500) (Invitrogen)
- Anti-rabbit Alexa647 (used 1:500) (Invitrogen)
Tissue preparation
Tissue preparation
Day 1: Whole mouse brain (2 years old, male) was fixed overnight in 4%PFA.
Day 2: Brain was washed 3x 5' in PBS + 4% Sucrose
Day2: Brain was incubated overnight in PBS + 30% Sucrose
Day3: Brain was embedded into optimal cutting medium (OCT)
Day3: Brain was cut with a cryostat in 10um thick sections and stored at -80C
Antibody staining protocol day 1
Antibody staining protocol day 1
15m
15m
Slides with brain tissue sections were thawed 00:15:00 Room temperature
15m
Add slides to glass coplin Jar.
Wash 3x 00:10:00 in PEMTx
10m
Move slides to plastic coplin jar for antigen retrieval
Perform antigen retrieval 00:15:00 85 °C
15m
Let plastic coplin jar cool down to room temperature 00:20:00
20m
Place slides back in original glass complin jar
Wash 2x 00:10:00 in PEMTx
10m
Place slides horizontally in box humidified with MQ. Use hydrophobic pen to outline tissue sections.
Add blocking buffer to the slides and incubate 01:00:00
1h
Add antibody solution (blocking buffer + 1:100 LBP110 + 1:100 p62) and incubate Overnight 4 °C
Antibody staining protocol day 2
Antibody staining protocol day 2
15m
15m
Wash slides 4x 00:15:00
15m
Incubate with secondary antibody solution in humidified box (PEMTx + 2% BSA + 1:500 secondary antibodies + 1:1000 DAPI) for 01:00:00
1h
Place slides back in coplin jar (keep in dark)
Wash 3x 00:10:00 in PEMTx
10m
Wash 2x 00:07:00 in PEM
7m
Mount slides using Prolong Gold mounting medium and store at 4 °C
Protocol references
LBP110 antibody: Kibbey, M. C., Jucker, M., Weeks, B. S., Neve, R. L., van Nostrandi, W. E., & Kleinman, H. K. (1993). f8-Amyloid precursor protein binds to the neurite-promoting IKVAV site of laminin. In Proc. Natl. Acad. Sci. USA (Vol. 90).