Labeling of Biocytin-Filled Interneurons in Rat Hippocampal Slices for Visualization

Abstract

Source: Economides, G., et al. Biocytin Recovery and 3D Reconstructions of Filled Hippocampal CA2 Interneurons. J. Vis. Exp. (2018)

This video demonstrates the procedure for staining fixed rat brain slices containing biocytin-filled interneurons. The protocol includes incubation with avidin-biotin complex (ABC), staining with HRP and chromogenic substrate, preserving structures with osmium tetroxide, and embedding the tissue in resin for imaging.

Protocol

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

1. Determination of Calcium Binding Protein or Protein Content of Interneurons and Biocytin Visualization Following Electrophysiological Recordings and Biocytin Filling

Note: At the end of the electrophysiological recordings, slices that contain biocytin-filled cells are fixed overnight prior to histological procedures. The fixative solution will be replaced with a single change of 0.1 M phosphate buffer the next morning if the rest of the procedure is carried out on another day in order to prevent tissue damage. The fixation solution (4% paraformaldehyde, 0.2% saturated picric acid solution, 0.025% glutaraldehyde solution in 0.1 M phosphate buffer (PB)) must be made fresh on the day of the recordings for best results.

1. At the end of the electrophysiological recording, carefully pick up the slice containing the recorded cell(s) with a paintbrush and place it in a pot containing artificial cerebral spinal fluid (ACSF).
   NOTE: Details of the protocol used for slice preparation and intracellular recordings using sharp electrodes have been described previously.
2. In a fume hood, carefully pick up the brain slice with a paintbrush and roll it onto a small piece of fine-quality filter paper. Place another piece of moistened filter paper onto the slice and place the two pieces of wet filter paper in a small plastic pot containing 5-10 mL of fixative solution and store in the fridge overnight at 4 °C.
3. Prepare a solution of gelatin in distilled water. Place 20 mL of distilled water in a beaker on a hot plate and heat it to 60 °C. Add progressively 2.4 g of gelatin to the water, wait until dissolved, and allow it to cool to 35–40 °C before use to prevent tissue damage.
4. Replace the fixative solution with 2 mL of 0.1 M PB. Place the slice in a Petri dish and cut away any excess tissue with a scalpel blade. Place the trimmed tissue in a Petri dish (diameter of 9 cm, height of 1.4 cm), ensuring that it lies flat with no folds or creases, and remove the excess buffer using a dry paintbrush.
5. Cover the tissue with the warm gelatin solution and place the Petri dish onto a frozen block to quickly cool the solution.
6. Using an angle-poise lamp to provide contrast, look through the side of the dish and keep the slice flat using light pressure from a fine paintbrush until the gelatin begins to set.
   NOTE: The gelatin can be re-melted if the tissue does not lie flat. Any imperfections on the surface of the gelatin caused by the removal of the paintbrush as it solidifies may be removed by melting the surface layer gently by moving the bulb of the lamp close to the surface for a few seconds.
7. Move the dish of setting gelatin to the fridge and leave at 4 °C for 30-60 min.
8. In a fume hood, cut out a small block (~ 1 x 1 cm) containing the gelatin-embedded tissue of the dish using a scalpel blade, lift the block using a small spatula, and carefully place it in the same but fresh fixative solution used to fix the slices for at least 30 min at 4 °C.
9. Wash the gelatin block in 5 mL of 0.1 M PB three times, dry it using a piece of paper tissue, and stick the block side up (i.e., with the tissue at the top) onto a vibratome chuck using super glue.
10. Remove excess glue with a piece of filter paper and use a scalpel blade to cut the corners of the block off, leaving a diamond shape.
11. Section the slice at 50 µm thickness using a vibratome and place each section carefully in a glass vial containing 10% sucrose.
12. Carefully pick up a section from the vial, and place it flat into a Petri dish lid. Using a dissecting microscope and a fresh scalpel blade, remove the gelatin from around the section and return the section to a vial containing 2 mL of fresh 10% sucrose
    NOTE: It is crucial to remove as much gelatin as possible at this stage to reduce tissue shrinkage during the dehydration step (Step 1.37).
13. Cryo-protect the sections in 0.1 M PB-based sucrose-glycerol solution at room temperature by incubating them for 10 min in 10% sucrose solution, 20 min in 20% sucrose-6% glycerol solution twice, and finally 30 min in 30% sucrose-12% glycerol solution twice under constant agitation.
14. Place the sections flat onto a small rectangle of tin foil using a paintbrush. Remove any excess liquid from the sections and carefully fold the tin foil into a parcel.
15. Hold the parcel close to the surface of liquid nitrogen without touching the surface for 30 s and then allow the sections to thaw completely for approximately 30 s. Repeat the freeze-thaw another two times.
16. Remove all sections with a paintbrush and place them in a glass vial containing 2 mL of 0.1 M PB under constant agitation to wash off excess sucrose.
17. Remove the PB with a Pasteur pipette and incubate the sections in 2 mL of 1% aqueous hydrogen peroxide (H₂O₂) for 30 min. Wash the sections in 2 mL of 0.1 M PB 3x 5 min.
18. Remove the 0.1 M PB with a Pasteur pipette and add 1 % sodium borohydride (NaBH₄) in 0.1 M PB.
    NOTE: Do not cap the vial, as NaBH₄ solution gives off hydrogen gas.
19. Remove the sodium borohydride with a Pasteur pipette and wash the sections thoroughly in 2 mL of 0.1 M PB 5x 5 min.
20. Replace the 0.1 M PB with 10% normal goat serum (NGS) in 0.1 M PB for 30 min.
21. Remove the goat serum and incubate the sections overnight at 4 °C in a mixture of mouse monoclonal and rabbit polyclonal antibodies made up in ABC solution.
    NOTE: A list of primary antibodies used in previous studies is displayed in Table 1.
22. Incubate the sections for 2 h in the dark in a mixture of fluorescently labeled secondary antibodies (Table of Materials).
23. Mount the sections onto the slides in mounting medium and cover them with a coverslip.
24. Take images of fluorescence labeling at 40X magnification (Figure 1Bb).
25. After the fluorescence imaging, place the slide into a glass Petri dish containing PBS and carefully remove the coverslip. Then wash the sections off the slide using gentle pulses of PBS from a Pasteur pipette. Place the sections into a clean glass vial containing PBS.
26. Perform the avidin-HRP reaction by firstly incubating the sections in ABC for at least 2 h to amplify the HRP reaction product.
27. Prepare the 3,5 diaminobenzidine (DAB) solution by adding one tablet to 5 mL of distilled water.
28. Wash the sections with PBS three times for 10 min and then with Tris buffer twice for 10 min. Remove the Tris buffer after the last wash.
29. Quickly add one drop of 8 % NiCl₂ (Nickel chloride) solution to the DAB solution, pipette the solution in and out to mix, and quickly add 1 mL of this solution over the sections. Incubate the sections in the DAB/NiCl₂ solution for 15 min.
30. Add 10 µL of 1% H₂O₂ to the DAB solution. Allow the reaction to proceed in the dark under constant agitation for about 1 to 2 min, and monitor the labeling of the filled cells with a dissecting microscope.
31. Stop the reaction by removing the DAB/NiCl₂/H₂O₂ solution and wash the sections with Tris buffer twice for 5 min.
32. In a fume hood, place a small circle of filter paper into a Petri dish and dampen it with 0.1 M PB. Lift the sections one at a time from the glass vial using a paintbrush, and place them carefully flat upon the paper.
33. Cover the sections with another moistened circle of filter paper and remove excess buffer by gently touching tissue paper to the surface.
34. Apply 8–9 drops of 1% osmium tetroxide in 0.1 M PB to the top paper, cover the dish, and retain in the fume hood for at least 30 min, but no more than 1 h.
35. Open the Petri dish and lift the top filter paper. Lift the sections carefully one at a time with a paintbrush, place them in a glass vial, and rinse them in distilled water twice.
36. Dispose of osmium tetroxide waste appropriately. Rinse all disposable equipment and place them in appropriate bins.
37. Place each section flat onto a glass slide and coverslip the sections. Transfer the slide into a Petri dish, place an empty glass vial over the coverslip to retain it in place, and cover with 50% alcohol. After 15 min, remove the slide from the solution and remove the sections from the slide. Place the sections back on the slide and then place the slide in 70% alcohol for 15 min. Repeat the same process with 95% and finally 100% alcohol solution.
38. Following the dehydration step, transfer the sections to a glass vial containing 100% alcohol on a shaker in a fume hood. Replace the alcohol solution with propylene oxide (C₃H₆O) and wash three times for 5 min. Following the last wash, keep ~2 mL of propylene oxide in the vial and add resin (1:1 ratio). Ensure that the resin is dissolved and keep the sections under constant agitation for 30 min.
39. Place each section in an aluminum planchette containing epoxy resin using a wooden stick and incubate overnight.
    NOTE: Do not leave the sections in the resin longer than 24 h to avoid the risk of damaging the sections.
40. Place the planchette over a hot plate for approximately 10 min. Pick up each section with a wooden stick and place them on a clean slide. Keep the orientation of each section consistent using a dissecting microscope. Place a coverslip over the sections. Place the slide in the oven for 48 h at 56 °C for curing.

Table 1: Table of solutions.

Solutions used	Composition/Instructions
Fixation solution	4% paraformaldehyde, 0.2% saturated picric acid solution, 0.025% glutaraldehyde solution in 0.1 M phosphate buffer (PB)
0.1M Phosphate Buffer pH 7.6	Add 100 mL of stock 1 M Phosphate Buffer to 900 mL of distilled water
Phosphate buffered saline (PBS) pH 7.4/7.5	Add 10 mL of 0.1M phosphate buffer, 0.2 g of KCl, and 8.76 g of NaCl to 990 mL of distilled water
TRIS buffer pH 7.5	Dissolve 5.72 g of Tris Hydrochloride and 1.66 g of Tris Base in 50 mL of distilled water. Then make up to 1 L with distilled water.
Buffered glutaraldehyde and paraformaldehyde fixative solution	4% paraformaldehyde, 0.2% saturated picric acid solution, 0.025% glutaraldehyde solution in 0.1 M Phosphate buffer.
ABC solution	Solution to be made at least 30 min before use from the ABC kit. Add 1 drop of solution A and 1 drop of solution B to 2.5 mL of PBS.
Durcupan epoxy resin:	To make 20 pots: 20 g of component A, 20 g of component B, 0.6 g of component C and 0.4 g of component D-
	Protect the balance from spills by covering the plate with a circle of filter paper. Carefully weigh the reagents into a tripour beaker in the proportions stated above. Mix thoroughly by vigorously stirring using two wooden sticks for at least 5 min. The mixture should become a uniform density dark brown color. Place the beaker into the oven at ~50 °C for a maximum of 10 min to remove as many air bubbles as possible. NOTE: The resin will start to cure if you leave the beaker in the oven longer than 10 min. Decant the resin out into plastic pots or 5 mL syringes, date them, and store them in the -20 °C freezer ready for use.

Results

Figure 1: Neuronal reconstructions of two types of basket cells recorded and filled in the hippocampal CA2 region and correlated electrophysiological data obtained following intracellular recordings in vitro. This figure has b...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Avidin-7-amino-4-methylcoumarin-3-acetic acid (Avidin-AMCA) antibody- 20.8 mg/mL	Vector laboratories	A2008
Biocytin ≥98% (TLC)	Sigma	B4261
3,5 diaminobenzidine (DAB) tablet, To prepare 5 mL	Sigma	D4293
Durcupan epoxy resin A	Sigma	44611
Durcupan epoxy resin B	Sigma	44612
Durcupan epoxy resin C	Sigma	44613
Durcupan epoxy resin D	Sigma	44614
Ethanol, puriss. p.a., absolute, ≥99.8%	Sigma	32221
Gelatin	Sigma	48723
Glutaraldehyde solution, grade I, 25 % in H2O	Sigma	G5882
Glycerol, ≥99%	Sigma	G5516
Goat serum	Sigma	G9023
Goat anti-mouse fluorescein isothiocyanate (FITC)- 14.3 mg/mL	Sigma	F2653
Goat anti-rabbit Texas Red (TR)- 3.3 mg/mL	Invitrogen	T2767
Hydrogen peroxide, 30% solution	Sigma	H-1009
Immersion oil, viscosity 1.250 cSt (lit.)	Sigma	I0890
Nickel chloride	Sigma	N5756
Osmium tetroxide, for electron microscopy, 4% in water	Sigma	75632
Paraformaldehyde, reagent grade, crystalline	Sigma	P6148
Picric acid, moistened with water, ≥98%	Sigma	197378
Phosphate buffer 1 M	Sigma	P3619
Propylene oxide, 99%	Alfa Aesar	30765
Sodium tetrahydroborate	VWR	27885.134
Sucrose	Fisher scientific	S/8600/53
Trizma Hydrochloride, ≥99.0%	Sigma	T5941
Trizma base, ≥99.9%	Sigma	T6066
Vectashield Antifade Mounting Medium, , refractive index 1.45	Vector laboratories	H-1000
Vectastain Elite ABC HRP kit	Vector laboratories	PK6100
Equipment used
Vibratome	Agar Scientific
C4A Cupped aluminium planchettes	GA-MA & ASSOCIATES, INC.
Leica DMR microscope	Leica Microsystems
X-Cite 120PC Q fluorescence light source	Excelitas Technologies
Leica DFC450 digital microscope camera	Leica Microsystems
Rapidograph technical drawing pen 0.18mm	London graphics centre
0.18mm rapidograph nib	London graphics centre
Rapidograph technical drawing pen 0.25mm	London graphics centre
0.25mm rapidograph nib	London graphics centre
Neurolucida system including PC workstation, stage, camera and joystic for XYZ stage control	Microbrighfield (MBF) Bioscience
Neurolucida software version 2017	Microbrighfield (MBF) Bioscience
CorelDRAW graphics Suite X5	Corel
Video editing software	Adobe Premiere Pro
Glass vials 14 mL	Fisher scientific