High-Resolution Episcopic Microscopy (HREM) system performing block-face imaging on a 15 mm embedded tissue sample, capturing micron-resolution images after each microtome cut.

HREM Protocol

Protocol for Performing HREM 3D Imaging

Consumables

Essential

37 ℃ water bath
PBSA or calcium-free Hanks saline
Fine forceps (#5)
Fine scissors (blade length no more than 1 cm)
Stereomicroscope KCl stock solution (for example, 1 M)

Desirable

Fine iridectomy scissors (blade length of a few mm)
37 ℃ heat pad
Heparin

Procedure

Working in PBSA or calcium-free Hanks saline at 37 ℃, remove embryos from the yolk sac.
- The addition of heparin to the PBSA or saline at this stage can help minimise blood clotting.
Maintain embryos at 37 ℃ using a water bath or dry heat pad.
Monitor using a stereomicroscope, flipping embryos periodically to minimise the pooling of blood on one side.
Repeatedly clip the umbilical vessels to maintain blood flow for as long as possible.
When the blood flow ceases, transfer the embryos to saline supplemented with at least 50 mM KCl.

This step increases the probability that the heart arrests in diastole

Where the identification of cardiovascular phenotypes is the main reason for carrying out HREM imaging, it is recommended that the heart is isolated from the embryo. The isolation process depends on the stage of the embryo, and it should always be followed by blood removal.

Consumables

Essential

37 ℃ water bath
PBSA or calcium‐free Hanks saline
Fine forceps (#5)
Fine scissors (blade length no more than 1 cm)
KCl stock solution (for example, 1 M)
4 % PFA

Desirable

Fine iridectomy scissors (blade length of a few mm)
Heparin

Embryos E11.5 and older

Working in PBSA or calcium-free Hank’s saline at 37 ℃, decapitate the embryo immediately below the lower jaw.
- Do not cut any lower, to avoid slicing through the great vessels.
- The addition of heparin to the PBSA or saline at this stage can help minimise blood clotting.
Let the embryo bleed out for ~5 min at 37 ℃ until no more blood is lost.
- Trim the umbilical vessels occasionally if blood clots block them.
- Elevated potassium levels at this stage (for example, 50 mM KCl) will then increase the probability that the heart arrests in diastole once blood flow has ceased.
Cut transversely at, or below, the liver to remove the lower portion of the torso.
- Do not cut above the liver or you risk damaging the heart.
Using fine scissors (or iridectomy scissors) cut down the ventral midline from the head end.
- Keep the point of the scissors angled outwards and slightly diagonally towards a lower limb. This will cut through the ribs, allowing the chest cavity to be opened up to reveal the heart.
- Gently pulling the forearms apart will help to achieve this.
Pull with forceps or cut with iridectomy scissors along the edges of the diaphragm, releasing it from attachment to the sides of the body wall.
Using forceps, gently ease the diaphragm and/or aorta upwards (ventrally, towards the head) whilst holding the dorsal torso down with forceps.
- This should pull the entire assembly of heart, pericardium, lungs and thymus out of the chest cavity.
- The dissection can also be done with portions of intestine and/or liver still attached.
Break the superior caval veins to release the assembly from the body.
Remove at least one of the lung lobes.
- This will help with blood removal at a later stage.
Transfer the dissected organs into 4 % PFA for 20–30 minutes.
- Ensure that the sample does not float on the surface. Surface tension will permanently distort the atria and ventricles.
- A crude dissection is sufficient at this stage, leaving the lungs, thymus and diaphragm attached if necessary.
- Do not fix the embryos for longer than 30 minutes, or blood lysis will become difficult.

The process of blood removal should then be started immediately.

Embryos E11.5 and older

Working in PBSA or calcium-free Hank’s saline at 37 ℃, decapitate the embryo immediately below the lower jaw.
- Do not cut any lower, to avoid slicing through the great vessels.
- The addition of heparin to the PBSA or saline at this stage can help minimise blood clotting.
Let the embryo bleed out for ~5 min at 37 ℃ until no more blood is lost.
- Trim the umbilical vessels occasionally if blood clots block them.
- Elevated potassium levels at this stage (for example, 50 mM KCl) will then increase the probability that the heart arrests in diastole once blood flow has ceased.
Cut transversely at, or below, the liver to remove the lower portion of the torso.
- Do not cut above the liver or you risk damaging the heart.
Using fine scissors (or iridectomy scissors) cut down the ventral midline from the head end.
- Keep the point of the scissors angled outwards and slightly diagonally towards a lower limb. This will cut through the ribs, allowing the chest cavity to be opened to reveal the heart.
- Gently pulling the forearms apart will help to achieve this.
Pull with forceps or cut with iridectomy scissors along the edges of the diaphragm, releasing it from attachment to the sides of the body wall.
Using forceps, gently ease the diaphragm and/or aorta upwards (ventrally, towards the head) whilst holding the dorsal torso down with forceps.
- This should pull the entire assembly of heart, pericardium, lungs and thymus out of the chest cavity.
- The dissection can also be done with portions of intestine and/or liver still attached.
Break the superior caval veins to release the assembly from the body.
Remove at least one of the lung lobes.
- This will help with blood removal at a later stage.
Transfer the dissected organs into 4 % PFA for 20–30 minutes.
- Ensure that the sample does not float on the surface. Surface tension will permanently distort the atria and ventricles.
- A crude dissection is sufficient at this stage, leaving the lungs, thymus and diaphragm attached if necessary.
- Do not fix the embryos for longer than 30 minutes, or blood lysis will become difficult.

The process of blood removal should then be started immediately.

Newborn Pups

The lungs of newborn pups contain air. This causes the dissected tissue to float, making dissection more difficult.

Working in PBSA or calcium‐free Hank’s saline at 37 ℃, decapitate the embryo immediately below the lower jaw.
- Do not cut any lower, to avoid slicing through the great vessels.
- The addition of heparin to the PBSA or saline at this stage can help minimise blood clotting.
- Elevated potassium levels at this stage (for example, 50 mM KCl) increase the probability that the heart arrests in diastole once blood flow has ceased.
Let the embryo bleed out for ~5 min at 37 ℃ until no more blood is lost.
- Trim the umbilical vessels occasionally if blood clots block them.
Cut transversely at, or below, the liver to remove the lower portion of the torso.
- Do not cut above the liver to avoid damaging the heart.
Using fine scissors (or iridectomy scissors) cut down the ventral midline from the head end.
- Keep the point of the scissors angled outwards and slightly diagonally towards a lower limb. This will cut through the ribs, allowing the chest cavity to be opened up to reveal the heart.
- Gently pulling the forearms apart will help to achieve this.
Whilst the heart remains within the thorax, carefully remove the lung lobes using forceps or iridectomy scissors.
Pull with forceps or cut with iridectomy scissors along the edges of the diaphragm, releasing it from attachment to the sides of the body wall.
Allow the heart to bleed out for a few minutes.
Using forceps, gently ease the diaphragm and/or aorta upwards (ventrally, towards the head) whilst holding the dorsal torso down with forceps.
- This should pull the entire assembly of heart, pericardium, lungs and thymus out of the chest cavity.
Break the superior caval veins to release the assembly from the body.
Transfer the dissected organs into 4 % PFA at room temperature for 20–30 minutes.
- Ensure that the sample does not float on the surface. Surface tension will permanently distort the atria and ventricles.
- A crude dissection is sufficient at this stage, leaving the lungs, thymus and diaphragm attached if necessary.
- Do not fix the embryos for longer than 30 minutes, or blood lysis will become difficult.

The process of blood removal should then be started immediately

Embryos Younger Than E11.5

Carefully expose the heart by removing at least some of the pericardium.
- Take care not to exert any pressure to pull upwards on the head/pharyngeal region. This will damage the distal outflow tract and aortic sac connections
Decapitate the embryo above the lower jaw/pharyngeal region.
Cut transversely at or below the liver to remove lower portion of torso.
- Do not cut above the liver.
Fix the torsos for 30 minutes in fresh 4 % PFA at room temperature.
- Ensure that the sample does not float on the surface. Surface tension will permanently distort the atria and ventricles.
- Do not fix the embryos for longer than 30 minutes, or blood lysis will become difficult.

The process of blood removal should then be started immediately

Blood removal dramatically improves the quality of HREM images of the heart. For maximum effectiveness the procedure should be carried out immediately after dissection, without allowing the embryos to cool. The required timings can vary depending on the precise batch of fix, so all steps should be monitored using a dissecting microscope and the timings adjusted if necessary.

Consumables

4 % PFA
Distilled or tap water

Embryos E11.5 or, Newborn pups

Once the samples have been fixed for 20–30 minutes at room temperature, wash extensively in water to remove the PFA.
Place the sample in a large volume of water on gentle roller bars for up to 60 minutes to lyse the blood. Change the water at least 3 or 4 times during this time. Use a 15 ml tube for E11.5 embryos, a 50 ml tube for late gestation or neonatal samples. Check the appearance of the heart regularly. Alternatively, samples can be lysed individually in a 6‐well dish, agitated on a rocker or rotating platform.
End the water treatment as soon as blood lysis has occurred. Hearts will appear translucent white/faint pink. Dark, punctate blood clots should be largely or completely replaced by a faint pink tinge from blood lysis. Water treatment longer than ~ 60 minutes can cause structural damage. For example, E13.5–15.5 hearts split along the plane of the ventricular wall. This can cause the appearance of a blister, generally on the right ventricle, below the outflow.
Transfer back to 4 % PFA for overnight fixation.

Samples are now ready for fine dissection prior to preparation for embedding. Careful removal of the thymus will reveal the aorta and pulmonary trunk. Removal of the pericardium will reveal the atria, while removal of the lungs will expose the ventricles.

Embryos Younger Than E11.5

Once the samples have been fixed for 30 minutes at room temperature, wash extensively in distilled water to remove the PFA.
Place in a fresh volume of water and agitate for up to 60 minutes. A 15 ml tube on gentle roller bars would be sufficient for a batch of embryos. A 12‐well microtitre plate on a vigorous rocker or orbital skier platform would be sufficient for individual embryos.
Wash the sample when most, if not all, of the blood has lysed.
Transfer to 4 % PFA for overnight fixation.

In preparation for embedding and imaging, samples are dehydrated then infiltrated with a JB-4 dye mix. The exact procedure depends on the size and tissue density of the sample. The procedure below is intended as a guide, but the parameters are empirical, and some experimentation may be required with your own samples.

Consumables

Consumables JB-4 resin embedding kit
- Polysciences JB-4 Embedding Kit containing Solution A (monomer), Solution B (accelerator) and Catalyst (Benzoyl Peroxide, Plasticised).
Eosin B
- Sigma Aldrich Eosin B
Acridine Orange
- Sigma Aldrich Acridine Orange
Filters
- Sartorius 0.22μm polyethersulfone (PES) membrane filter

Final Volume	Solution A (ml)	Eosin B (g)	Catalyst (g)	Acridine Orange (g)
300	300	0.825	3.75	0.169
200	200	0.55	2.5	0.113
150	150	0.413	1.88	0.084
100	100	0.275	1.25	0.056
80	80	0.220	1.00	0.045

JB4 Dye Mix Preparation

Measure Solution A and place on a stirrer.
While mixing vigorously, add the catalyst slowly to avoid the formation of lumps.
Slowly add both dyes.
Stir for at least 4 hours (preferably overnight) at room temperature or below.
Filter the mix through 0.22 μm PES membrane to remove dust and any undissolved dye.
- There should be very little or no undissolved dye to remove. If there are lots of undissolved dye grains you may need to start again. The mix will break standard filter membranes.
- At this point, the JB-4 dye mix can be stored at 4 ℃ for 2-3 weeks. Any leftover mix can be used to prepare 50:50 JB-4/methanol for infiltration

Procedure

For an E14.5 mouse embryo (roughly 10mm by 5mm):

Dehydration

Fix the sample overnight in Bouin's fixative, then transfer to PBS.
- Other fixatives including 10 % formalin, 4 % paraformaldehyde and Dent's can be used. We prefer Bouin's fixative for whole embryos since it gives better preservation of the structure of soft mesenchymal tissue, which tends to collapse during dehydration.
Over the course of 1-2 days, wash samples with repeated changes of PBS for 2 hours per wash.
Dehydrate samples using a MeOH series (10 %, 20 %, 30 %, 40% 50 %, 60 %, 70 %) for 2 hours per step.
If using Bouin's fixative, wash the sample in 70 % MeOH containing 1% ammonia for three minutes.
- This helps remove any residual yellow picric acid staining left from the Bouin's fix.
Complete dehydration using MeOH mixes (80 %, 90 %, 95 %, 100 %) for 2 hours each. Samples can be held overnight at any stage prior to 90 % if necessary.

Infiltration

Immerse the sample overnight in a 50:50 mix of MeOH:JB-4 dye mix.
Briefly rinse in JB-4 dye mix to help minimise residual MeOH, then immerse in several ml of fresh JB-4 dye mix.
Leave to infiltrate at 4 ℃, preferably with gentle rocking. For example, infiltration of an E14.5 embryo usually takes 3 or 4 nights.
The sample is now ready to be embedded.

For small embryos or embryo tissue pieces, the length of dehydration and infiltration steps can be reduced drastically:

For E9.5 embryos:
- Dehydration steps need only be 30-60 minutes and infiltration takes only 3-4 hours.
For denser embryo tissue such as an E14.5-E18.5 heart:
- Dehydrate in 1-hour steps and infiltrate overnight.
For larger or much more dense samples such as adult tissues:
- Successful infiltration can take more than a week and needs to be determined empirically

Following infiltration, samples are embedded in polymerised JB-4 dye mix

Consumables

JB-4 dye-mix, as prepared during the infiltration process.
Solution B as provided in the JB-4 resin embedding kit.

Equipment

A stereomicroscope with very bright illumination from the base.
- This enables samples to be visualised during the embedding procedure, despite the dark red colour of the JB-4 mix.
- As an example, the Schott KL 2500 LCD fibre optic lamp, with a 250-watt bulb, providing a large bundle diameter up to 15 mm.
Glass capillaries to manipulate samples, e.g. glass Pasteur pipette lengths that have been heated to produce blunt ends.
- Although fine forceps can be used to manipulate samples, there is less danger of damage using glass capillaries.
Moulds for embedding the samples.
- Block moulds are available at various sizes from Indigo Scientific. These are not listed on the website, but can be ordered by emailing hello@indigo-scientific.co.uk
Plastic chucks to attach to the base of the samples.
- Custom-made chucks are available from Indigo Scientific. These are not listed on the website, but can be ordered by emailing hello@indigo-scientific.co.uk
- If samples are to be attached directly using the JB-4 mix, it is best to use chucks with both a central hole and cross grooves. This is more convenient than using traditional 2-step procedures in which an adhesive resin is used to attach the polymerised block to the chuck.

After embedding, polymerised blocks are removed from moulds and trimmed. We recommend samples are baked to ensure optimal hardness. This stage is optional, but it can improve the stability of the sample during sectioning. Baking times are empirical, and some experimentation may be required with your own samples

Procedure

Invert the mould onto paper towels to remove mineral oil and unpolymerized JB-4 mix.
Check that polymerised JB-4 is visible in both the central hole of the chuck and through the base of the chuck.
- This is essential to avoid the sample block breaking off during sectioning.
- If necessary, a little fresh JB-4 embedding mix can be added to the top of the chucks and left to polymerise for a few hours. If the block does break off, use abrasive paper to ensure the surface of the chuck is completely flat (it is not flat from manufacture).
- Do the same with the base surface of the JB-4 block and then use SuperGlue contact adhesive to reattach it to the plastic chuck.
Remove blocks from the mould and wipe dry.
- For the small (5 mm) EMS flexible moulds, blocks can be popped out by firm pressure from underneath. Excess polymerised JB-4 must be removed from the side of the chuck using a blunt blade to enable the block to fit in the HREM block holder
- For the larger PTFE moulds, grip the plastic chuck with an adjustable wrench and pull each block vertically. Be careful not to apply too much pressure with the wrench - this can cause distortion of the chuck, weakening its attachment to the sample block.
- If the blocks are very soft and jelly-like or rubbery immediately after removal from the mould, there is likely to be something wrong with your JB-4 mix or its components. Normal blocks will sometimes feel slightly springy when removed from the mould, especially if they are large in size. This is usually fixed by baking.
Bake the samples at 95 to 100 ℃ to ensure the blocks are completely hard before sectioning.
- For small (5 mm) samples, 12 hours is sufficient.
- Larger blocks benefit from longer times. For E14.5 mouse embryos (14- 16 mm depth blocks), we bake for 24 to 48 hours. The blocks will become soft and pliable after 5 minutes of baking but harden rapidly after removal from the oven. Baking has no effect on the sample or subsequent image quality but baking for excessive periods can produce brittle blocks that can be prone to accidental chipping or cracking.
Cool the block in a fridge for several hours until completely hard.

It can be convenient to trim off much of the JB-4 cushion prior to HREM imaging. This can be carried out either on the HREM apparatus or using a manual microtome. Trimming can be performed either before or after baking and cooling of blocks. To measure the approximate cushion thickness, view the block on its side and illuminated from below in a bright light beam. Occasionally, blocks can break during trimming. Excessive baking can result in brittle blocks and these can sometimes shatter, especially if the section depth is unusually large. SuperGlue is very effective for repairing broken blocks.

Basic Embedding Procedure

At room temperature, add Solution B to cold JB-4 dye mix and mix vigorously. We recommend 0.6 ml of Solution B per 10 ml of JB-4 dye mix.
- Use glass scintillation vials, with up to 20 ml of mix per vial, and add twice the manufacturer’s recommendation of Solution B as the dyes appear to have some inhibitory effect on polymerisation.
Fill the mould with the polymerising mix and add the sample, ensuring no air bubbles are trapped on its surface.
Between approximately 10 and 30 minutes later, there will be an optimum period of a few minutes during which the viscosity of the mix increases. At this point, samples can be realigned and will be held in their new positions.
- If Solution B is added to a room temperature JB-4 mix, polymerisation will occur more quickly and there will only be a very brief period of increasing viscosity. If the mix does not polymerise fully, or is taking longer to polymerise, check your components by testing polymerisation of JB-4 without dyes. Incomplete removal of methanol during sample infiltration also appears to inhibit polymerisation.
Adjust samples as necessary, but avoid excessive movement as this can cause unsightly variations in the background of HREM images.
- Polymerisation proceeds from the bottom upwards. As a result, samples will be held more firmly at the bottom and can be easily damaged by realignments in very viscous mix.
Place a plastic chuck on top of the mould, ensuring that enough mixture is present to fill up the central hole in the chuck and the cross grooves.
- Note that only some of this will polymerise, so it’s essential that there is enough to attach the block adequately to the chuck.
Leave the samples to polymerise completely in an oxygen-free atmosphere. It is sufficient to cover the exposed surface of JB-4 with a thin layer of mineral oil.
Samples will polymerise in a couple of hours or less but are best left overnight at room temperature.

Samples embedded using this method will rest on the bottom of the mould (i.e. the top surface of the block). As a result, after polymerisation, the sample will often protrude slightly through the block surface, and this will be visible in the first HREM images.

Embedding With a Cushion

If it is important to obtain a complete image series (e.g. for complete 3D models), samples should be embedded after first forming a thin cushion of polymerised JB-4 at the base of the mould. (Since JB-4 polymerisation is oxygen sensitive, it is extremely difficult to polymerise a layer thinner than 1-2 mm).

Procedure

Add sufficient polymerising mix to the mould to obtain a layer 2-3 mm thick and completely cover with mineral oil.
Leave to polymerise for 30-60 minutes before following the Basic Embedding Procedure. (It is not necessary to remove the mineral oil from the cushion before proceeding).

Note: that the cushions will have a slightly convex surface, which can make stable positioning of samples difficult. One solution is to wait until increasing viscosity of the mix holds the sample in position. Alternatively, after filling the mould with polymerisation mix, but before adding the sample, it is possible to flip the cushion upside down with fine forceps. This ensures that there is a flat surface for the sample to rest on. Note also that cushion thickness may vary from sample to sample, despite identical polymerisation conditions.

The cushion will be visible in the raw HREM images, since the interface between the cushion and the remainder of the JB-4 block generally appears much brighter than the rest of the plastic. This effect appears to get worse, the longer the time gap between polymerisation of the cushion and embedding the sample. It is not, therefore, advisable to leave cushions overnight before use.

Contact our High-Resolution Episcopic Microscopy (HREM) Experts

Request a quote or a meeting to discuss the Optical HREM Micro instrument using the form.

Phone: +44(0) 1462633500

Email: hello@indigo-scientific.co.uk

HREM Protocol

Consumables

Essential

Desirable

Procedure

Consumables

Essential

Desirable

Embryos E11.5 and older

Embryos E11.5 and older

Newborn Pups

Embryos Younger Than E11.5

Consumables

Embryos E11.5 or, Newborn pups

Embryos Younger Than E11.5

Consumables

JB4 Dye Mix Preparation

Procedure

For an E14.5 mouse embryo (roughly 10mm by 5mm):

Dehydration

Infiltration

For small embryos or embryo tissue pieces, the length of dehydration and infiltration steps can be reduced drastically:

For E9.5 embryos:

For denser embryo tissue such as an E14.5-E18.5 heart:

For larger or much more dense samples such as adult tissues:

Consumables

Equipment

Procedure

Basic Embedding Procedure

Embedding With a Cushion

Procedure

Contact our High-Resolution Episcopic Microscopy (HREM) Experts

Request a quote or a meeting to discuss the Optical HREM Micro instrument using the form.

HREM

HREM Applications

Optical Products

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