Assessing Attractive or Repulsive Activity of Proteins Using Hippocampal Neurons

Abstract

Source: Yamagishi, S. et al., Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons. J. Vis. Exp. (2016)

This video demonstrates a method to assess protein activity using hippocampal neurons over patterned protein zones. Neurons exhibit repulsion in test protein zones and attachment in control protein zones, highlighting the effects of attractive or repulsive protein activity.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Matrices

1. Boil 4-8 silicone matrices in a microwave or on a hot plate for 5 min and allow them to dry completely for 1 h under laminar flow (striped side up).
   NOTE: The following procedures should be performed under laminar flow.
2. Blow compressed air or use transparent sticky tape to remove any dust from the striped side of the matrices. Keep the striped side clean so it can firmly adhere to the plate surface.
3. ....

Results

Figure 1. Silicon Matrix used to create the striped protein stamp. (A, B) Top view of the silicon matrix. The size of matrix is 30 mm x 25 mm x 5mm. White arrow in A indicates a small hole where the recombinant protein is injected (step 2.2). Arrowhead .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL centrifuge tube	Violamo	1-3500-01
Alexa Fluor 488 Goat anti-human IgG antibody	Thermo Scientific	A11013
Alexa Fluor 594 Donkey anti-mouse IgG antibody	Thermo Scientific	A-21203	Dilution 1/500
B27 supplement	Thermo Scientific	17504-044	Dilution 1/50
Bovine serum albumin	Sigma	01-2030-2
Cell strainer 100 um	BD Falcon	352360
Centrifugation machine	Kubota	2410
DAKO pen	DAKO	S2002	Alternative water-repellent pen may be used
Disposable scalpel	Feather	2975#11
FBS	Thermo Scientific	10437-028
Forceps No. 5	Fine Science Tools	11254-20
GlutaMAX	Thermo Scientific	35050-061	Dilution 1/200
Hamilton Syringe	Hamilton	805N	22 gauge, 50 uL
HBSS	Thermo Scientific	14170-112
Human IgG, Fc Fragment	Jackson	009-000-008
Laminin	Thermo Scientific	23017-015
Neurobasal	Thermo Scientific	21103-049
PBS	Nacalai	14249-24
Penicillin-Streptomycin	Thermo Scientific	15070-063	Dilution 1/100
Plastic culture dish, 60 mm	Thermo Scientific	150288
Silicone Matrices			Available and purchasable from Prof. Martin Bastmeyer (bastmeyer@kit.edu)
Stereo Microscope	Olympus	SZ61
Tip, 1000 uL	Watson	125-1000S
Transparent sticky tape	Tesa	57315	Alternative sticky tape may be used
Trypan blue, 0.4%	Bio-Rad	145-0013
Trypsin/EDTA	Thermo Scientific	25300-054
Culture medium			Neurobasal supplemented with B27, GlutaMAX and Penicillin-Streptomycin.