Immunostaining of Interneurons in a Rat Hippocampal Section

Abstract

Swietek, B., et al., Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers. J. Vis. Exp. (2016)

This video demonstrates the process of visualizing a biocytin-filled interneuron in a rat hippocampal section, highlighting its neurochemical identity through the use of specific antibodies and fluorescence microscopy.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Staining of the Primary Antibody and Biocytin

(Day 1)
NOTE: The following immunostaining procedure is for free-floating sections and requires continuous shaking at a low speed (2 revolutions/min, rpm) on a shaker for all incubation steps.

1. Wash the tissue 3x for 10 min each in 0.1 M PBS (pH = 7.4).
   NOTE: 0.1 M PBS can be commercially purchased or made in the laboratory as follows (makes 1 L): 8 g of NaCl, 0.2 g of....

Results

Figure 1. Suggested Plane for Approaching Cells for Biocytin Fills. The image illustrates a Granule Cell (GC) filled with biocytin during recording that has been processed to reveal biocytin (in red using 594-conjugated streptavidin). The GC has it.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaCl	Sigma	S7653	Immunostaining
KCL	Fluka	60129	Immunostaining
Disodium hydrogen phosphate	Sigma	S7907	Immunostaining
Monopotassium phosphate	Sigma	229806	Immunostaining
Triton X-100	Sigma	T8787	Immunostaining
Guinea pig anti CB1	Sigma	Af530-1	Immunostaining
Streptavidin, Alexa Fluor conjugate	Molecular Probes	S11227	Immunostaining
Normal goat serum	Sigma	G9023	Immunostaining
Vectashield	Vector Labs	H-1000	Immunostaining
Secondary Antibodies	Invitogen Molecular probes	Alexa Fluor conjugated dyes	Immunostaining
Labnet orbit low speed shaker	Bioexpress	S-2030-LS	Immunostaining
Forceps	Dumont	11231-30	Immunostaining
Slide folders	EMS	71520	Immunostaining
Biocytin	Sigma	B4261	Electrophysiology