Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Summary

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

Abstract

Despite all the technological advances at the light microscopy level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Introduction

Light microscopy, including confocal and two-photon microscopy, has proven to be an efficient tool for studying in vivo neuronal processes, among other things^1,2. Although the typical spatial resolution at the Light Microscopic (LM) level is approximately 200 nm, recent technological advances using different light sources, such as extreme ultraviolet and soft X-ray microscopy, have notably increased this resolution to a nearly 10 nm spatial resolution^3,4,5. Other technological advances in imaging include combined magnetic resonance imaging with histology and provide a novel method for measuring the thickness of the myelin sheath in vivo, a parameter that was traditionally measurable only at the Electron Microscopic (EM) level^6,7. Although these advances at the LM level provide an excellent tool for studying living processes, a detailed view and characterization of structures, such as synaptic contacts, can only be achieved with EM, which offers a resolution that can reach 0.5 nm. However, observation at the EM level requires the specimens to be dead and altered in some ways, with chemical fixatives and dehydration processes, in order to preserve the cytoarchitecture. Thus, examining biological samples at high resolution can be challenging due to radiation damage from the electron beam, low contrast, structural deviations of membranes, or even the presence of artifacts that can occur following dehydration and epoxy embedding^8,9,10.

Preserving specimens in their native form for structural analysis can be achieved by using "Cryo EM of Vitrified Sections" or CEMOVIS, a sectioning approach that involves rapidly freezing and embedding the sample in vitreous ice and examining the sections under the EM at a cryogenic temperature^11,12. This procedure allows for the examination of samples while they are still solid and fully hydrated, thus eliminating artifacts caused by dehydration processes¹³. However, this method involves additional devices for cryo-ultramicrotomy, as well as additional devices on the standard EM, in order to allow this observation at very low temperatures, which generate significant additional costs. In addition, the CEMOVIS approach precludes the use of immunolabeling techniques, since antibodies usually have to be incubated at RT. Alternatively, it is possible to combine ultrastructural analysis with immunohistochemical procedures by using a freeze-substitution approach, during which cryo-fixed specimens are slowly thawed while immerged in cryo-protective chemicals and are then embedded in specialized resins, such as Lowicryls. Post-embedding immunolabeling can then be performed on such material¹². However, freeze-substitution and cryo-fixation techniques are time consuming. They require the installation of additional equipment and still require samples to be exposed to organic solvent and chemical fixative that can alter the cytoarchitecture, despite the use of a low temperature^14,15. Hence, despite all the technological advances both at the LM and EM level, chemical fixation of brain tissue, particularly with acrolein, remains a low-cost and time-efficient method to combine immunohistochemistry with EM¹⁶.

In the last decades, many attempts were made to find a mixture of aldehydes that provide the best tissue preservation. Before the 1960s, the only chemical fixative that gave acceptable results for EM was osmium tetroxide. However, osmium tetroxide is highly toxic and expensive, precluding its use through the vascular system to fix organs such as the brain. Acrolein was introduced in the late 1950s as a reliable method for animal tissue preservation suitable for EM observation of cellular structures¹⁷. It penetrates the tissue more deeply and reacts more quickly than other aldehydes when used for fixation by immersion and allows good preservation of cytoplasmic components, with minimal shrinkage of the tissue¹⁷. Such a feature gives acrolein fixation a clear advantage over other aldehydes when used in fresh tissue, by allowing a more accurate localization of living molecular compounds, such as enzymes and other proteins¹⁸. Indeed, it has been validated through the years as an easy, efficient and low-cost method of fixation for visualization at the EM level in many species, including amphibians and rodents, as it efficiently stabilizes peptides and proteins, retains antigenicity and provides relatively intact ultrastructure when used in combination with another aldehyde fixative^{16,18,19,20,21}. Protocols for acrolein fixation in rodents have since then been standardized and used extensively, particularly by the Pickel group, to implement dual immunolabeling for EM^16,22. A few groups have used acrolein fixation in non-human primate brain tissue²³. However, to our knowledge, there is only one published protocol efficiently describing chemical fixation with acrolein in non-human primates that is compatible with EM immunolabeling²⁴.

In this article, we provide an easy and reliable method to efficiently chemically fix non-human primate brains with acrolein, allowing for a potentially long-term preservation along with pre-embedding immunolabeling and transmission EM examination.

Protocol

Ethics Statement: All protocols involving animals were approved by the Comité de Protection des Animaux de l'Université Laval and were made in accordance with the Canadian Council on Animal Care's Guide to the Care and Use of Experimental Animals (Ed. 2). The protocol described here was optimized for adult animals of approximately 800 g. The volumes of fixative should be adjusted according to the animal's size.

1. Preparation of Solutions for Transcardiac Perfusion

1. Prepare 1 L of a 50 mM sodium Phosphate-buffered Saline (PBS) solution according to the following steps. Prepare the solution at most 24 h before perfusion and keep at 4 °C until use.
   1. Measure 800 mL of distilled water in a 1 L beaker. Add 5.87 g of dibasic anhydrous sodium phosphate (Na₂HPO₄), 1.20 g of monobasic monohydrate sodium phosphate (NaH₂PO₄·H₂O), and 9 g of sodium chloride (NaCl). Stir to dissolve.
   2. Adjust the pH to 7.4 by gradually adding 5 N NaOH. Add distilled water to reach a total volume of 1 L.
      CAUTION: NaOH is a corrosive chemical compound. Wear the appropriate Personal Protective Equipment (PPE; laboratory coat, gloves, protective goggles, etc.).
2. Prepare 2 L of 4% paraformaldehyde (PFA) according to the following steps. This solution should be prepared at most 24 h before surgery and kept at 4 °C until use.
   1. Measure 1.5 L of distilled water in a 2-L beaker. Add 23.48 g of dibasic anhydrous sodium phosphate (Na₂HPO₄) and 4.80 g of monobasic monohydrate sodium phosphate (NaH₂PO₄·H₂O). Stir to dissolve. Add distilled water to reach a total volume of 2 L.
   2. Heat the solution under a venting hood until the temperature reaches approximately 45 - 55 °C. Do not heat to over 60 °C.
   3. Gradually add 80 g of PFA to the solution and stir until completely dissolved (approximately 30 - 60 min). Keep monitoring the temperature to keep it below 60 °C.
      CAUTION: PFA is highly volatile in its powder form. It is highly toxic if in contact with eyes or skin and is hazardous in case of inhalation or ingestion. Wear PPE and use with caution under a venting hood.
   4. Cool the solution down to 4 °C and filter. Store at 4 °C.
3. Prepare 1 L of 3% acrolein in 0.1 M Phosphate Buffer (PB) according to the following steps. The PB solution should be prepared at most 24 h prior to perfusion.
   CAUTION: Acrolein is highly toxic if inhaled and can produce immediate damage. It is also corrosive and highly toxic if absorbed through the skin. It is carcinogen and mutagenic. Wear the appropriate PPE and use under a venting hood.
   1. Measure 800 mL of distilled water. Add 8.66 g of dibasic anhydrous sodium phosphate (Na₂HPO₄) and 5.38 g of monobasic monohydrate sodium phosphate (NaH₂PO₄·H₂O). Stir until all the salts dissolve. Add distilled water to reach a total volume of 1 L. Keep the solution at 4 °C.
   2. Before surgery, transfer 900 mL of the PB solution to a 1 L glass container and add 30.94 mL of 97% acrolein solution under a venting hood. Add PB solution to reach a total volume of 1 L and stir. Filter the solution and keep it at 4 °C.

2. Transcardiac Perfusion and Brain Dissection

1. Keep solutions on ice during the entire surgical procedure. Prepare the pump by placing the tube in the first solution to be used (PBS; 50 mM) and turning the pump on until no air remains in the hose. For the transcardiac perfusion of a macaque monkey, use a 21 G needle and set the outflow at approximately 80 mL/min.
2. Anesthetize the animal with an intramuscular injection of a mixture of ketamine (20 mg/kg), xylazine (4 mg/kg), and acepromazine (0.5 mg/kg). Maintain the animal under isoflurane (3%) sedation.
3. Attach the animal's limbs to a venting table.
4. With a scalpel, remove the skin up to the armpits. Cut the abdominal muscles. Use heavy-duty surgical scissors to cut the ribs laterally by carefully avoiding the vital organs.
5. Cut the diaphragm with surgical scissors and raise the rib cage to expose the heart. Once the diaphragm is cut, proceed quickly, since the heart will stop beating within minutes.
6. Remove the pericardium with a scalpel blade and insert the needle into the left ventricle. With a scalpel, carefully make a small excision to the right atrium. Quickly start the pump at 72 mL/min and hold the needle in place. If possible, tilt the animal in order to have its head on a lower level than the heart.
   NOTE: Be careful not to pierce the interventricular septum when inserting the needle.
   1. Rinse the blood with approximately 300 mL of PBS until the lungs are white and no blood comes out of the right atrium.
7. Stop the pump, quickly transfer the hose/tube to the 3% acrolein solution, and start the pump again. Gradually increase the pumping speed to 80 mL/min once the heart has stopped beating. Perfuse approximately 500 mL of the acrolein solution.
8. Stop the pump, quickly transfer the hose to the 4% PFA solution, and start the pump again. This step requires approximately 1 L of PFA.
   NOTE: The fixation is complete when the forelimbs are rigid and the neck is stiff.
9. Cut the head and carefully dissect the brain out of the skull. Take care to not damage the brain with the surgical instruments. The perfusion is optimal when the brain is pale (no trace of blood) and rigid (Figure 1A).
10. Immerse the intact brain in 4% PFA for 1 h at 4 °C.
11. Serially cut the brain with a cooling vibratome (4 °C) in the desired plane into 50 µm thick sections and collect them in PBS (0.1 M) (Figure 1B).
    NOTE: This step can be carried out in many ways according to the desired protocol. Hemispheres can be separated prior to vibratome cutting, or kept whole. If the brain is too large for the vibratome platform, it can be cut into smaller blocks. After this step, sections can be stored for a long period of time at -30 °C in an antifreeze solution made of 40% PB (50 mM), 30% glycerol, and 30% ethylene glycol.

3. Pre-embedding Immunohistochemistry (Figure 1C)

1. Prepare a 4 L stock solution of Tris-buffered Saline (TBS, 50 mM, pH 7.6) as follows.
   1. Measure 2L of distilled water in a 4 L beaker, add 24.23 g of trihydroxymethyl aminomethane (THAM; C₄H₁₁NO₃), and stir to dissolve.
   2. Adjust the pH to 7.6 with approximately 148 mL of 1 N HCl. The acid should be added with caution to avoid reaching a pH below 7.6. The total volume should be 4 L.
      CAUTION: HCl is highly corrosive. Wear the appropriate PPE.
2. Select sections (from step 2.11) containing the region of interest to be processed for EM immunohistochemistry.
3. Wash the free-floating sections 3x in PBS (0.1 M, pH 7.4) for 5 min at RT to rinse the antifreeze solution.
4. Prepare a 0.5% solution of NaBH₄ diluted in PBS.
   1. Weigh 0.05 g of NaBH₄ and dilute it in 10 mL of PBS. Do not cover. Prepare this solution just before use.
5. Incubate the sections in the freshly prepared NaBH₄ solution for 30 min at RT. Rock gently. Do not cover.
6. Wash 3x in PBS for 10 min at RT, rocking vigorously until none of the reaction gas remains.
7. Prepare a blocking solution for EM with 2% appropriate normal serum and 0.5% cold fish gelatin diluted in PBS.
   NOTE: The quantity should be calculated in order to have enough for the following three steps. Use a serum made from the same animal species hosting the secondary antibody. Avoid the use of antigen retrieval methods or addition of small amounts of triton (to increase the penetration of antibodies), as these methods significantly compromise the quality of the tissue.
8. Incubate the sections in the blocking solution for 1 h at RT. Rock gently.
9. Prepare primary antibody solution diluted in the blocking solution.
   NOTE: The concentration of the primary antibody is usually the same as for LM immunohistochemistry, but consider conducting tests with different antibody concentrations beforehand, as some primary antibodies may not work with acrolein. If so, it is possible to use a mix of glutaraldehyde (0.1 - 2%) and 4% PFA for transcardiac perfusion and obtain similar results. Optimize the incubation time and temperature as well.
10. Incubate the sections in primary antibody solution overnight at RT with gentle rocking. Use a previously tested incubation time and temperature.
11. Wash the sections 3x in PBS for 5 min at RT with gentle rocking.
12. Prepare a 1:1,000 solution of biotinylated secondary antibody diluted in the blocking solution.
    NOTE: The secondary antibody must be raised against the host species used to generate the primary antibody.
13. Incubate the sections in secondary antibody solution for 1.5 h at RT. Rock gently.
14. Prepare an avidin-biotin-peroxidase (ABC) solution at least 60 min prior to the end of the secondary antibody incubation.
    1. Use a calibrated pipette to measure 8.80 µL/mL of solutions A and B and dilute them in PBS.
    2. Rock mildly for at least 60 min at RT to allow the complete binding between the avidin and biotin molecules.
15. After the incubation in secondary antibody solution, wash 3x in PBS for 10 min at RT.
16. Incubate the sections in the ABC solution for 1 h at RT. Rock gently.
17. Wash once in PBS and twice in TBS for 10 min at RT with gentle rocking.
18. Prepare a fresh solution of 0.05% 3,3'-diaminobenzidine (DAB) with 0.005% H₂O₂ diluted in TBS.
    1. Weigh 12.5 mg of DAB and dilute it in 25 mL of cold TBS. Protect from light.
       CAUTION: The DAB powder is highly volatile and harmful if inhaled. It is carcinogenic and teratogenic. Thus, pregnant or nursing women should not manipulate this product, even when diluted. Use a N95 mask when manipulating and wear PPE.
    2. Filter the solution and add 4.5 µL of 30% H₂O₂ just before use.
19. Incubate sections in the DAB solution for 3 to 7 min at RT. Rock gently.
    NOTE: The brown precipitate should not be too dark in order to avoid a high level of background staining. The incubation time should be optimized accordingly.
20. Stop the reaction by quickly washing twice in cold TBS, then twice for 10 min in cold TBS at RT, followed by twice for 10 min in PB, with mild rocking.
    NOTE: The use of PB (and not PBS) is critical in order to eliminate any traces of NaCl, as it would react with osmium and form crystals.

4. Osmification and Embedding for Electron Microscopic Observation

1. Prepare a solution of 1% osmium tetroxide (OsO₄) diluted in PB. Protect from light.
   CAUTION: Osmium is highly toxic and should not come in contact with the skin, eyes, or mouth and should not be inhaled. It can cause death if ingested. It should only be used under a venting hood and with appropriate PPE.
2. Incubate the sections in OsO₄ solution for 30 min at RT under the venting hood (without agitation) and cover them with aluminum foil to protect them from light. Completely flatten the sections prior to adding the OsO₄ solution.
   NOTE: The sections become very dark and rigid and should be manipulated with care following this step.
3. Prepare water-repellent epoxy resin during the osmification.
   1. Add appropriate amounts of each component of the epoxy resin mix (20 g of epoxy resin, 20 g of hardener, 0.6 g of accelerator and 0.4 g of plasticizer) to a large plastic cup. Stir with a wooden stick or plastic pipette until a homogeneous brown color is obtained.
      NOTE: It is critical to use the exact proportion of each component.
   2. Transfer equal quantities to aluminum cups of appropriate sizes, depending on the number of sections to be processed. Allow it to rest.
4. Wash the osmificated sections 3x in PB for 10 min at RT with low-speed rocking.
5. Dehydrate the sections in the following series of graded ethanol for 2 min each: 2 times in 35% ethanol; 1 time each in 50, 70, 80, 90, and 95% ethanol; and 3x in 100% ethanol.
6. Transfer the sections to glass vials to complete the dehydration process by incubating the sections 3 times for 2 min in propylene oxide.
   CAUTION: Propylene oxide is a highly volatile and toxic organic solvent. It can cause serious damage in the eyes or skin in case of contact or if inhaled or ingested. It has been classified as a grade 2 carcinogenic substance. It should only be used under a venting hood and with PPE. It is also highly flammable and should be kept away from any heat source.
   NOTE: Prior to this step, sections should be carefully transferred in glass vials, since propylene oxide is an organic solvent and is incompatible with plastic. At this point, sections are very fragile and should be manipulated with care. Sections can be alternatively transferred to glass vials prior to step 4.5.
7. Transfer sections carefully, one by one, in the aluminum cups and avoid contact with air as much as possible. Flat-embed the sections in previously mixed water-repellent epoxy resin and incubate them overnight under the venting hood at RT.
   NOTE: At this step, the sections are completely dehydrated and very fragile and should be manipulated with care.
8. Using mineral oil, prepare grease-coated glass slides along with greased plastic coverslips.
9. Soften the resin by incubating aluminum cups at 60 °C for 12-15 min at most. Carefully flatten the sections on the greased side of the glass slide. Place the greased coverslip and carefully push out any remaining air.
10. Incubate the slides at 60 °C for 48 h.
    NOTE: It is critical to not exceed 48 h incubation time, as the resin will become too hard.
11. Remove the plastic coverslip.

5. Sample Preparation for Ultrathin Sectioning and Observation Using a Transmission Electron Microscope

1. Use binoculars to find the region of interest and cut a small quadrangular piece of approximately 1 mm² with a scalpel.
2. File the tip of a resin block and glue the quadrangular piece onto it (Figure 1C). Allow the glue to dry for at least 1 h or overnight prior to sectioning.
3. Using an ultramicrotome, cut the quadrangular piece into 80 µm-thick sections (Figure 1D).
   1. Place the resin block in an ultramicrotome deck in the vertical position and, using a sharp razor blade, gradually cut each side of the resin block to form a trapezoid with smooth sides.
   2. Put the deck in its horizontal position and rotate the block until the longest side of the trapezoid is facing downward.
   3. Use a diamond trimming tool or a glass knife to trim the surface of the quadrangular piece. Set the ultramicrotome to cut 300 µm thick sections at 1 mm/s. Adjust the knife to be vertically parallel to the quadrangular piece and to display a very small horizontal angle of approximately 1°.
      NOTE: This angle will allow the user to approach the tissue almost parallel to the surface of the block, where immunolabeled elements are more likely to be found. When using these parameters with the diamond trimming tool, the resin displays a shiny white color. When the tissue is being cut, it changes to a purple or greenish color. 
   4. Use an ultra 45° diamond knife equipped with a boat filled with distilled water to cut 80 µm thick sections, smoothen sections by passing over them with a piece of absorbing paper tipped in xylene, and collect serial sections on formvar-coated nickel slot grids or bare 150 mesh copper grids (Figure 1E).
4. Place the grids in a grid storage box.
5. Stain the grids with lead citrate.
   1. Use a 5 mL syringe and a 0.2 µm syringe filter to prepare a 1:1 solution of filtered lead citrate stock solution and filtered distilled water. Protect it from light.
      NOTE: The stock solution of lead citrate should be made fresh every month to avoid the formation of solid deposits. See the material data sheet for the stock recipe. In addition, if many series of grids need to be stained, change the diluted solution when it becomes milky.
   2. Place each grid onto a drop of the diluted solution, with the section in contact with the solution. Incubate for 3 min.
   3. Use small tweezers to hold the grid, and thoroughly rinse it in two beakers containing distilled water.
   4. Remove excess water by gently using absorbing paper. Store the grids in a grid box. Wait 30 min before examining the sections by Transmission Electron Microscopy (TEM; Figure 1F).

Results

In this section, we present representative results that were obtained following the observation, at the transmission EM level, of immunostained primate brain tissue chemically fixed with a mixture of 3% acrolein and 4% PFA. We achieved good preservation of the ultrastructure, as indicated by the relatively intact myelin sheath and the neat visualization of double membranes (Figure 2A). Synaptic contacts, along with neuronal elements from the microenvironment, can easily b...

Discussion

In this article, we present a reliable protocol for transcardiac perfusion of non-human primates and pre-embedding immunohistochemistry suitable for EM sample examination. Although typical cryo-EM, such as CEMOVIS, provides a good preservation of brain ultrastructure, it also limits the use of immunohistochemistry¹². Other techniques, including cryo-substitution and Tokuyaso technique, allow post-embedding immunohistochemistry, but these techniques are expensive due to additional devices needed du...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dibasic anhydrous sodium phosphate (Na2HPO4)	Fisher scientific	S374-500
Monobasic monohydrate sodium phosphate (NaH2PO4·H2O)	EM Science	SX0710-1
Sodium chloride (NaCl)	Fisher scientific	S271-3
Hydroxymethyl aminomethane (THAM)	Fisher scientific	T370-500
HCl	EMD	HX0603-3	1 N dilution. Product is corrosive. Use with appropriate protection.
NaOH	EMD	SX0590-1	5 N dilution. Product is corrosive. Use with appropriate protection.
Paraformaldehyde (PFA)	Sigma	P6148	4% dilution. Product is highly volatile in its powder form and highly toxic. Use with caution under a venting hood with appropriate protection.
Acrolein (90%)	Sigma	110221	3% dilution. Product is highly toxic. Use under a venting hood with appropriate protection.
Autopsy venting table	Mopec	CE400
Electronic perfusion pump	cole parmer	masterflex L/S 7523-90
Needle (perfusion)	terumo	NN-1838R	18 G 11/2
Needle	terumo	NN-2713R	21 G 1/2
Ketamine			20 mg/kg
Xylazine			4 mg/kg
Acepromazine			0.5 mg/kg
Scalpel
Scalpel blades	Feather           lance	201011           J9913	No.22 for surgery and No. 11 for EM
Surgical scissors
Rongeurs
Vibratome	Leica	VT 1200S	Calibrate blade before each use, when the device allows it
Vibratome razor blade	Gillette	GIN 642107
Glycerol	Fisher scientific	G33-4	30% dilution
Ethylene glycol	Fisher scientific	E178-4	30% dilution
Sodium borohydride (NaBH4)	Sigma	S-9125
Normal horse serum (NHS)	Jackson immunoResearch Laboratories	008-000-121	2% dilution
Cold-fish gelatin	Aurion	900.033	0.5% dilution. Original product is concentrated at 40%
Primary antibody, SERT	Santa Cruz biotechnology	SC-1458	1/500 dilution
Primary antibody, ChAT	Chemicon (Millipore)	AB144P	1/25 dilution
Primary antibody, TH	ImmunoStar	22941	1/1,000 dilution
Biotinylated secondary antibody, goat	Vector laboratories	BA-9500	1/1,000 dilution
Biotinylated secondary antibody, mouse	Vector laboratories	BA-2000	1/1,000 dilution
Vectastain elite ABC kit	Vector laboratories	PK6100	8.8 µL/mL of A and B each
3,3'-diaminobenzidine (DAB)	Sigma	D5637	0.05% dilution. Product is highly volatile in its powder form and toxic. Do not throw waste in the sink.
Peroxide (H2O2) 30%	Fisher scientific	H-323	0.005% dilution
Osmium tetroxide (OsO4)	Electron microscopic science	2% 19152 4% 19150	Original solution can be either 2 or 4%. Keep attention to which one is used to calculate the final 1% dilution. Product is very sensitive to light. Osmium is highly toxic. Use only under a venting hood with appropriate protection.
Durcupan water-repellent epoxy resin	Sigma	A: M epoxy resin (44611) B: hardener 964 (44612) C: accelerator 960 (DY 060) (44613) D: plasticizer (44614)	Polymerize 48 h at 58 °C before throwing in waste.
Alumium cups	Electron microscopic science	70048-01
Ethanol	commercial alcohols	1019C	Dilute in distilled water with appropriate concentration
Propylene oxide	Electron microscopic science	20401	Organic solvent. Highly volatile and toxic. Use under a venting hood.
Non-coated medium glass slides	brain research laboratories	3875-FR	Grease surface with mineral oil
Plastic film (Aclar embedding film)	Electron microscopic science	50425-25	Grease surface with mineral oil
Ultramicrotome	Leica UC7	EM UC7
Diamond trimming tool (ultratrim)	Diatome	UT 1081	Can use glass knife alternatively
Ultra 45° Diatome Diamond knife	Diatome	MC13437	equipped with a boat
Xylenes	Fisher scientific	X5SK-4
150-mesh copper grids	Electron microscopic science	G150-cu
grid-box	Electron microscopic science	71138	Can store up to 100 grids
Sodium citrate	Anachemia	81983
Lead nitrate	Sigma	L-6258	Make a stock solution of lead citrate made of 1.33 g of lead nitrate and 1.76 g of sodium citrate diluted in 42 mL of preboiled and cooled distilled water to which 8 mL of 1 N NaOH are added after the conversion from lead nitrate to lead citrate is complete. pH should be approximately 12. Store solution in a hermetic plastic bottle and protect from light.
Syringe	terumo	SS-05L	5 mL
Syringe filter	Corning	431222	0.2 µm
Absorbing paper (bibulous paper)	Electron microscopic science	70086-1
Parafilm	Laboratory film	PM-999
Mineral oil	Sigma	M5904