Jul 16, 2023

Public workspaceImmunization with alpha-synuclein peptide

DOI
dx.doi.org/10.17504/protocols.io.e6nvwd5bwlmk/v1
  • 1Columbia University
Connor Monahan
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwd5bwlmk/v1
Protocol Citation: Connor Monahan 2023. Immunization with alpha-synuclein peptide. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwd5bwlmk/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 12, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84886
Keywords: T cell, immunization, emulsion, Complete Freund’s Adjuvant, EAE, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: 0375
Abstract
This protocol details immunization of MHC-II(-/-); HLA DRB1*15:01 mice with alpha-synuclein(32-46) peptide in complete Freund's adjuvant.
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Materials
Materials

ReagentBD Difco™ AdjuvantsFisher ScientificCatalog #DF0638-60-7

ReagentPertussis Toxin from B. pertussis Lyophilized (Salt-Free)List LabsCatalog ##181

Immunization
Immunization
Prepare immunization emulsions:
Prepare Amount200 µg of M. tuberculosis H37Ra (BD Difco Adjuvant, Fisher Scientific, Cat #DF0638-60-7; Waltham, MA) in 1x PBS or in Amount100 µg a-syn32-46 peptide (A&A peptides; San Diego, CA) dissolved in 1x PBS.
Immunize 8- to 12-week-old male and female mice subcutaneously with Amount50 µL of emulsion into the chest area of the mouse, near the axillary lymph node at both front limbs for a totally of Amount100 µL of emulsion injected per mouse.

The day of immunization is designated as day 0.
At day 14, boost each mouse with the same immunization dose at the same site.
Administer intravenous injections of Amount400 ng of B. pertussis toxin (List Biological Laboratories, Cat #181; Campbell, CA) at 14- and 16-days post-initial immunization (day 0 and 2 post boost).

Monitor mice daily for their weight, food and fluid intake and neurological deficits and their weights are recorded daily. Mice are classified as sick if they displayed 12% reduction from 0 DPI weight and/or hunched posture with ruffled and ungroomed fur.