Simultaneous Cryosectioning of Multiple Rodent Brains

Summary

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Abstract

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Introduction

Here, we present the protocol for a novel method, which we call Megabrain, developed to cryosection multiple rodent brains simultaneously for downstream immunohistochemical procedures. A Megabrain allows for the production of single slides containing tissue from multiple animals. This technique has been optimized to cut coronal sections from 9 adult rat hemispheres, or 5 adult full brains, simultaneously. Therefore, the technique is most applicable in large immunohistochemical studies or other analyses done on slide-mounted brain tissue from a large cohort of animals.

Immunohistochemistry involves the use of specific antibodies directed against proteins of interest to understand and characterize their expression and cellular changes in specific tissue^1,2,3. The use of immunohistochemistry is prevalent in neuroscience research, among other scientific disciplines, aiding in the cellular and molecular understanding of the brain⁴. Large-scale studies involving many animals and brain sections can be both resource and time intensive. As such, there is a multifaceted rationale behind the development of the Megabrain: to reduce time spent cryosectioning, mounting, and staining tissue, while consequently using less reagents. Moreover, the ability to streamline the process and stain multiple brains in the same round helps to alleviate some of the variability between staining batches, a limitation of immunohistochemistry³. In addition, sectioning a Megabrain is a time saving alternative to sectioning individual frozen rodent brains and allows for rapid comparison of tissue between animals or even treatment groups by microscopy techniques.

Protocol

The Megabrain technique has been optimized using whole and hemisected brains from male adult C57Bl6 mice⁵, juvenile Sprague-Dawley rats, and adult Sprague-Dawley rats that were transcardially perfused with 1x phosphate buffered saline (PBS) followed by 4% paraformaldehyde⁶. Similar outcomes can be accomplished in other strains of mouse and rat, in both sexes and at different ages. The study to generate data for this manuscript used juvenile Sprague-Dawley rats and was approved by the University of Arizona Institutional Animal Care and Use Committee, and experimental animals were cared for according the Guide For the Care and Use of Laboratory Animals⁷.

1. Cryoprotection of Perfused Brains

1. Following successful transcardial perfusion⁶, collect the full brain^6,8 from animals and post-fix by placing into a 25 mL vial of 4% paraformaldehyde in PBS for 24 h.
   CAUTION: Paraformaldehyde is a toxic tissue fixative; handle with care. Transfer paraformaldehyde into a specifically labeled waste container that identifies its concentration and volume inside a chemical fume hood and then store in the chemical waste cabinet until disposed by Chemical Safety or properly trained personnel.
2. After 24 h has elapsed, in a fume hood, remove brains from 4% paraformaldehyde using a spatula. Transfer the brain into a vial of approximately 20 mL of 15% sucrose solution in 1x Tris-buffered saline (TBS) until brain sinks to the bottom of the vial (approximately 24 h).
3. Following this, transfer the brain to a vial of approximately 20 mL of 30% sucrose solution in 1x TBS, until the brain sinks to the bottom of the vial (approximately 24–48 h).

2. Brain Freezing

1. Place a 500 mL glass beaker on a bed of dry ice in an ice bucket. Pour 300–400 mL of isopentane (2 methyl-butane) into the glass beaker.
2. Allow the temperature of the isopentane to reach between -45 °C and -50 °C. Closely monitor and maintain this temperature range with a thermometer, keeping it constant throughout the procedure. Ensure that temperature readings are taken from the middle of the solution, and not while touching the bottom or side of the beaker.
3. On the benchtop, fill a disposable embedding mold (see materials list) approximately 1/2 full of Optimal Cutting Temperature (OCT; see Table of Materials) Compound. Ensure that there are no air bubbles in the OCT. If there are any air bubbles remove them with a spatula or needle.
4. Remove the first brain from the vial of 30% sucrose with a spatula. At this point, either freeze brains whole or hemisect, depending on the study design.
   1. For hemisected brains, use a new razor blade to make a clean cut through the tissue to separate the hemispheres. If not required for the study, remove the cerebellum and olfactory bulbs at this stage. If the cerebellum is needed for the study, it is suggested to cut a flat area at the posterior end of the brain to aid the brain in standing straight in the mold. If the study only requires tissue from a localized region, a rodent brain matrix can be used to block the brain based on known coordinates.
5. Give brains a numeric naming system. Draw a diagram as shown in Figure 1. Write the number of the first brain to be placed in the Megabrain mold in position 2, leaving position 1 as a blank space to aid in quick identification of the orientation in which the brains were frozen.
6. Using blunt forceps or tweezers, pick up the brain to be placed in position 1. Orient the brain with the side to be cut first facing upwards. Lower the brain into the OCT in the appropriate location, using the tweezers to adjust its position until it stands independently.
7. Repeat steps 2.3–2.5 for the remaining brains/hemispheres to be frozen in positions 3–10. Avoid positioning brains/hemispheres in a symmetrical layout.
8. Review all 9 brains in the Megabrain mold. Once the brains are standing independently, upright, and correctly oriented in the OCT (as shown in Figure 2B), add more OCT to the mold (until the top of the brains are covered). Again, ensure that there are no visible air bubbles in the OCT.
9. Use the forceps to hold the corner of the mold, ensuring the forceps do not become partially submerged in the OCT (Figure 3A). Being careful to keep the mold level, lower the mold into the isopentane (-45 °C to -50°C) so that the bottom third of the mold is submerged. Hold it here to allow any bubbles in the OCT to rise to the surface and away from the tissue (Figure 3B).
10. After 30 s, lower the entire mold into the isopentane and release from the forceps, leaving the Megabrain submerged for at least 3 min (Figure 3C). Ensure that mold is kept level, so brains remain upright and equidistant (Figure 3D).
11. After 3 min, use the forceps to remove the mold containing the frozen, OCT embedded brains (Figure 3E) from the isopentane.
12. Immediately, wrap the mold and its contents in aluminum foil and label it with the animal numbers or Megabrain identification. Touch the frozen Megabrain as sparingly as possible to avoid thawing the tissue.
13. This Megabrain can now be transferred to a -20 °C freezer and stored for a minimum of 24 h.
    NOTE: Protocol can be paused here and the Megabrain can be stored frozen until cryosectioning takes place.

3. Preparing the Megabrain for Sectioning on the Cryostat

1. Set the cabinet temperature of the cryostat to -19 °C. Make sure this temperature is reached before continuing. Throughout sectioning, ensure that the cabinet temperature remains between -18 °C and -20 °C.
2. Take the Megabrain from the -20 °C freezer and place it into the cryostat. Also, place a chuck of the appropriate size dimensions, and two thin tipped paintbrushes into the cryostat. Leave both the Megabrain and the chuck in the cryostat for at least 15 min to come to the temperature of the cryostat.
3. Appropriately label slides with the Megabrain identification number and the slide number. Lay these slides on a slide warmer (35–45 °C).
4. Unwrap the Megabrain from the aluminum foil. Use a razor blade to vertically cut the corners of the mold to allow easy removal of the block of OCT from the mold (Figure 4A).
5. Dispense a thin layer (approximately 3 mm thick) of OCT onto the chuck.
6. Quickly, with minimal contact between fingers and the OCT, place the Megabrain on the chuck, in the orientation desired. Large forceps can also be used for this step to minimize thawing. Before starting to section, leave the Megabrain mounted chuck in the cryostat for 15–20 min to allow the OCT to freeze completely.
7. Once the base layer of OCT has frozen, apply a 2 mm thick layer OCT around the sides of the Megabrain and allow this to run down from the sides onto the chuck. This helps secure the Megabrain to the chuck. Again, before continuing, leave the Megabrain mounted chuck in the cryostat for 15–20 min to allow the OCT to freeze.
8. Use a razor blade to remove any excess OCT from the sides or bottom of the chuck (Figure 4B).

4. Cryosectioning the Megabrain

1. Before starting, ensure that a beaker containing at least 20 mL 1x PBS is accessible.
2. Position the chuck, mounted with the Megabrain, into the chuck head on the cryostat (Figure 4B).
3. Set the cryostat to cut at the desired thickness. The current methodology has been optimized for 10 µm to 50 µm sections.
4. Trim the OCT and tissue as needed to reach the desired brain region for tissue collection (Figure 4C).
5. When ready to collect tissue, lower the anti-roll plate until it rests on the stage and turn the cryostat handle to take a single section. Slowly lift off the anti-roll plate, being careful that the sectioned Megabrain is not attached to the anti-roll plate and does not fall off the stage Figure 4D).
6. Gently use the paintbrushes to unroll the Megabrain section until it is lying flat on the cryostat stage (Figure 5A). (If needed, hold the OCT flat using the paintbrushes to prevent rolling).
7. Remove slide from slide warmer and hover the slide, label-side down, over the Megabrain section on the stage allowing the section to stick to the slide.
8. Quickly apply a drop of 1x PBS to each tissue section on the slide and do not allow these to dry out until they have been brushed flat (see step 4.9).
9. Use a fine tipped paintbrush dipped in 1x PBS to brush out any bubbles in the tissue and unfold the tissue so it is lying flat on the slide (Figure 5B). Take care not to change the position of the brain sections and thus lose the relative position to the other tissue sections. Manipulate the tissue with care to avoid tears. Keep brain sections away from the edges of the slide, as peripheral space is required for a coverslip and PAP pen application in some staining protocols.
10. Place the slide back onto the slide warmer and dry for 45 min. Cover as needed to prevent dust from settling on the tissue. Once dry, store the slides in at -80 °C until further use.

Results

A positive end result to this procedure is tissue that lies flat on the slide, with no bubbles or tears, in the orientation in which they were frozen. Tissue sections are evenly spaced apart and easily identifiable due to good placement of the brain in the OCT and good notation as demonstrated in Figure 1. Assuming that the brain tissue was collected from animals of a similar age, and that the tissue was properly aligned in the OCT, sections collected on a sl...

Discussion

It should be considered during this procedure that the temperature of the Megabrain and its surroundings must be constantly monitored to prevent thawing and re-freezing of the tissue. The brain can only be removed from the -20 °C freezer up to 3 times as every time it is touched and left in the cryostat, with a warmer fluctuating temperature, the tissue thaws and refreezes, causing a jelly like texture and abnormal tissue integrity¹⁰. Therefore, it is optimal to cut the Megabrain all at once....

Materials

Name	Company	Catalog Number	Comments
Andwin scientific tissue-tek CRYO-OCT compound (case of 12)	Fisher Scientific	14-373-65
Thermo Scientific Shandon Peel-A-Way Disposable Embedding Molds	Fisher Scientific	18-41
Fisherbrand High Precision Straight Broad Strong Point Tweezers/Forceps	Fisher Scientific	12-000-128
Fisherbrand 20 mL HDPE Scintillation Vials with Polypropylene Cap	Fisher Scientific	03-337-23
Sucrose, poly bottle 2.5 kg	Fisher Scientific	S2-212	Both 15% and 30% sucrose concentrations need to be made up.
2-Methylbutane (Certified), Fisher Chemical	Fisher Scientific	O3551-4
PYREX Tall-Form Beakers	Fisher Scientific	02-546E
Fisherbrand General Purpose Liquid-in-Glass Partial Immersion Thermometers (-50 to +50 °C)	Fisher Scientific	13-201-642
Fisherbrand Scoopula Spatula	Fisher Scientific	14-357Q
STANLEY Razor Blade	Grainger	4A807
Edge-Rite Microtome blades	Fisher Scientific	14-070-60
Microscope slides (1" frost) - white	Fisher Scientific	22-034-979
Gibco PBS (Phosphate Buffered Saline) 10x, pH 7.2	Fisher Scientific	70-013-032	Dilute to 1X  before use
15 piece fine paint brushes	Amazon	B079J12ZRV
PAP pen	abcam	ab2601
Chuck Shown in Figure 4 was custom made by a lab technician, however similar sizes are available to order from other companies commercially.	Electron Microscopy Sciences	EMS065