Aug 30, 2024

Public workspaceImmunofluorescence for pSer129 alpha-synuclein and total alpha-synuclein in mouse brain 

DOI
dx.doi.org/10.17504/protocols.io.5jyl82819l2w/v1
  • 1University of Alabama at Birmingham
Laura Volpicelli-Daley
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DOI: dx.doi.org/10.17504/protocols.io.5jyl82819l2w/v1
Protocol Citation: Laura Volpicelli-Daley, Marissa Menard 2024. Immunofluorescence for pSer129 alpha-synuclein and total alpha-synuclein in mouse brain . protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82819l2w/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 19, 2024
Last Modified: August 30, 2024
Protocol Integer ID: 106252
Keywords: Immunofluorescence, Brain sections, Buffers, Incubation
Funders Acknowledgement:
Aligning Science Across Parkinson's Disease
Grant ID: 020616
Abstract
This protocol details the immunofluorescence of brain section for phopho-serine129-alpha-synuclein and total alpha-synuclein
Materials
Buffers:

Sodium Citrate Buffer (500mL)

AB
Sodium citrate1.47g
MQ H2O500mL
Adjust to 6.0 pH with 1N HCl
Tween-200.25 mL
store at 4oC

10X Tris buffered saline (TBS) (1L)
AB
Tris base24.22g
Sodium Chloride87.66 g
pH to 7.4 with hydrochloric acid
adjust to 1L with milliQ water
mix well. Autoclave or filter solution through 0.2µm filter into sterile bottle. Store at room temp.
1X Tris buffered saline (500 mL)
AB
10X TBS50 mL
milliQ water450 mL
Store at room temp
Other reagents:

ReagentRecombinant Anti-Alpha-synuclein (phospho S129) antibodyAbcamCatalog #ab51253
ReagentAnti-alpha + beta Synuclein antibody [EP1646Y]AbcamCatalog #ab51252
ReagentGoat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488Invitrogen - Thermo FisherCatalog #A-11034
ReagentDAPI and Hoechst Nucleic Acid StainsInvitrogen - Thermo FisherCatalog #H1399
Goat serum (Fisher NC9678224)
ReagentThermo Scientific Chemicals Triton™ X-100, 98%, for molecular biology, DNAse, RNAse and Protease freFisher ScientificCatalog #AC327371000 Dilute to 10% w/v in TBS. Store at 4°C.
ReagentProLong™ Gold Antifade MountantInvitrogen - Thermo FisherCatalog #P36934

Materials:

  • Ice in ice bucket
  • 6 well plates (non cell culture treated, Fisher 08-772-49; or the cheapest you can find).
-for rinses, blocking, and secondary antibody incubations 6 well plates can be re-used. After each use, clean with soapy water and scrub brush, rinse and let air dry.
-for primary antibody incubations, use new 6 well plate.
  • Squirt bottles to easily add 1X TBS to wells.
  • 15 mL Falcon tubes for blocking buffer, primary and secondary antibodies.
-for blocking, and secondary antibody incubations Falcon tubes can be re-used. After each use, clean with soapy water and scrub brush, rinse and let air dry.
-for primary antibodies, use new Falcon tube.

  • Vortex
  • ReagentFisherbrand™ Superfrost™ Plus Microscope SlidesFisher ScientificCatalog #12-550-15
  • 20, 200, 1000 µL pipetteman
  • Glass hooks for transferring sections
  • Fine paintbrush for mounting sections
  • Orbital shaker

  • netwell inserts (Fisher Scientific 07-200-213)ReagentCorning™ Costar™ Netwell™ InsertsFisher ScientificCatalog #07-200-213



Day 1
Day 1
2h 15m
2h 15m
Pre-warm sodium citrate buffer to Temperature37 °C .

Temperature
Place sections in netwell inserts (Fisher Scientific 07-200-213) in 6-well plate.
Wash 3X in cold 1X TBS for 5 min each with shaking (around 2 mL per well).
Wash
Mix
Wash in cold 1X TBS for Duration00:05:00 (1/3).

5m
Wash
Wash in cold 1X TBS for Duration00:05:00 (2/3).
5m
Wash
Wash in cold 1X TBS for Duration00:05:00 (3/3).
5m
Wash
Incubate at Temperature37 °C for Duration01:00:00 in pre-heated sodium citrate buffer (about 2 mL/well).

1h
Incubation
Temperature
Wash 3X in 1X TBS for 5 min each.
Wash
Wash in 1X TBS for Duration00:05:00 (1/3).

5m
Wash
Wash in 1X TBS for Duration00:05:00 (2/3).

5m
Wash
Wash in 1X TBS for Duration00:05:00 (3/3).

5m
Wash
Block for Duration01:00:00 at Temperature4 °C on shaker. Two mL of blocking buffer per well. Make sure sections are not folded on themselves.

ABC
Blocking buffer10mL Block (for example)
5% Normal Goat Serum→500µL Normal Goat Serum
0.1% Triton-X-100→100µL 10% Triton-X
1X TBS→9.4mL 1X TBS

1h
Temperature
Place in primary antibody solution DurationOvernight at Temperature4 °C , shaking. For sections that cover an entire mouse forebbrain with Amount40 undetermined sections spaced Amount240 undetermined apart we use Amount2 mL per well. If you are just using a couple sections, you can use Amount0.5 mL per well in a new 24 well plate. Make sure sections are not folded on themselves.


Note
  • Primary antibodies are diluted in 1X TBS and 5% goat serum
** NO MESH WELLS **
**USE NEW PLATES FOR INCUBATION**

  • Note catalog number and lot number (if available) in your notebook
  • For pS129-α-synuclein (Abcam, ab51253) dilute 1:5000. Note that more concentrated antibody results in higher nonspecific background. It is possible to use at a more dilute concentration, but would need to be determined by user.
  • For α+β synuclein (Abcam, ab51252) dilute 1:2500
  • Can combine with other primary antibodies that were not generated in rabbit.

1h
Mix
Overnight
Temperature
Day 2
Day 2
15m
15m
Wash 3X in cold 1X TBS for 5 min each with shaking.
Wash
Mix
Wash in cold 1X TBS for Duration00:05:00 with shaking (1/3).
5m
Wash
Wash in cold 1X TBS for Duration00:05:00 with shaking (2/3).
5m
Wash
Wash in cold 1X TBS for Duration00:05:00 with shaking (3/3).
5m
Wash
Place in secondary antibodies for Duration02:00:00 at Temperature4 °C , shaking. Cover plate with foil to protect from light.


Note
  • Secondary antibodies are diluted in 1X TBS and 5% goat serum
** NO MESH WELLS for incubation**
  • Use goat anti-rabbit Alexa 488 1:500 (Thermofisher A-11034)
  • Hoechst 33342 (Thermofisher H1399) also diluted 1:500

2h
Temperature
Wash 3X in 1X cold TBS for 5min each with shaking.
Wash
Wash in cold 1X TBS for Duration00:05:00 with shaking (1/3).
5m
Wash
Wash in cold 1X TBS for Duration00:05:00 with shaking (2/3).
5m
Wash
Wash in cold 1X TBS for Duration00:05:00 with shaking (3/3).
5m
Wash
Gently mount onto Superfrost plus slides in 1X TBS with a couple drops of 10% Tx-100.

Note
  • Try your best not to rip sections or allow sections to fold.
  • If you have sections representing entire brain, mount in order from rostral to caudal.
  • Make sure the coverslip will cover all the sections. If you don’t think it will cover the sections, use another slide. It’s better to mount fewer sections and make sure the coverslip will cover them.

Allow to dry, coverslip using ProlongGold.

Note
  • Pipette ProlongGold onto each section (about 30-50 µL).
  • Slowly lower coverslip over sections to prevent bubbles.

Pipetting
Protocol references
Mahoney-CraneCL, Viswanathan M, Russell D, Curtiss RAC, Freire J, Bobba SS, Coyle SD, Kandebo M, Yao L, Wan B-L, Hatcher NG, Smith SM, Marcus JN, Volpicelli-Daley LA.* (2023) Neuronopathic GBA1L444P mutation accelerates glucosylsphingosine levels and formation of hippocampal alpha-synuclein inclusions.  Journal of Neuroscience 43:501-421.