Using En Bloc Method to Increase Contrast

Safety Note:

Most of chemicals used for processing specimens for electron microscopy are extremely hazardous, especially glutaraldehyde, formaldehyde, osmium tetroxide, embedding medium in liquid form, lead citrate, uranyl acetate. Extreme care should be taken when handling these chemicals. All steps must be performed in a fume hood and gloves should be worn all the time. Osmium tetroxide, glutaraldehyde, propylene oxide/resin waste should be collected in bottles for safe disposal.

Specimen Preparation:

Tissue specimen should be cut into 1 mm pieces/blocks. Bigger specimen will result in incomplete infiltration therefore interfering final quality of results.

Fixation:

1. Fix tissue blocks in 4% formaldehyde and 1% glutaraldehyde in 0.1 M Phosphate Buffer (PB) (pH 7.4) for at least 2 hours at room temperature or overnight at 4 ºC.
2. Wash in 0.1 M PB 3x15min or overnight.
3. Post-fix in 1% osmium tetroxide in 0.1 M PB 1-2 hours at room temperature (Mix equal quantities of 2% aqueous OsO4 and 0.2M PB and use immediately).

En Bloc Staining (Enhancing Contrast):

1. Wash at least 3-5 times of 10 minutes each in distilled water to remove all excess phosphate ions therefore preventing uranyl acetate (UA) from being precipitated.
2. En bloc stain with 2% aqueous uranyl acetate for 1 hour at room temperature or 2 hours at 4 ºC in dark to prevent uranyl acetate from being precipitated as UA is photo reductive.
3. Wash 2-3 times of 5 minutes each in distilled water.

Dehydration:

1. 50% ethanol 10-15min
2. 70% Ethanol 10-15min (May be stored overnight at this stage if absolutely necessary)
3. 95% Ethanol 10-15min
4. 100% Ethanol 3x15min
5. 100% Propylene oxide 3x15min
6. 1:1 EMBed 812 and Propylene Oxide overnight at room temperature in tightly capped vials to prevent moisture from coming into specimen. It will be best if this step can be done on shaker.
7. Straight (100%) EMBed 812 for 1-2 hour at room temperature. Remove caps from vials to allow any remaining propylene oxide to evaporate.

Embedding:

1. Embed in beam capsules or embedding molds and label each sample.
2. Polymerise in 60 – 70 ºC (~65 ºC) oven for 24-48 hours.

Sectioning:  

1. Cut thick sections (0.5-1.0 um), and use a steel loop to transfer sections to a drop of water (this will flatten sections to avoid wrinkles - very important!!!) on slide and dry it on slide warmer or lamp. Toluidine blue stain for 1-3 min.
2. Observe sections under microscope for precise location to cut for ultrathin sections
3. Ultrathin sections at 60-90 nm thick (yellow-brown color) and collect sections onto grids, which stored in grids box. Allow grids to air dry for at least 2 hours or overnight before staining.

Staining:

1. Stain grids with uranyl acetate for 15-30 minutes and lead citrate for 3-15 minutes.
2. Observe under electron microscope.

Reagents:

0.2M PB, pH 7.4:

Na₂HPO4 ------------------------- 21.8 g

NaH₂PO4 ------------------------- 6.4 g

Distilled water ------------------ 1000 ml

4F1G Fixative (4% Formaldehyde & 1% Glutaraldehyde in 0.1 M PB, pH 7.4):

0.1M PB, pH 7.4 --------------- 88 ml

37-40% Formaldehyde --------- 10 ml

50% Glutaraldehyde ------------ 2 ml

1% Osmium in 0.1M PB:

2% Aqueous OsO4 -------------- 5 ml

0.2M PB, pH7.4 ----------------- 5 ml

5% Uranyl Acetate Solution:

To prepare 50 ml, add 2.5 g of uranyl acetate to 50 ml of distilled water. Cover with foil and stir overnight. Add 10 drops of glacial acetic acid. Store at 4 ºC. This solution is stable for at least 6 months at 4 ºC.

Reynold’s Lead Citrate Solution:

To prepare 50 ml, add chemicals in distilled water in following order

Lead nitrate -------------------------------- 1.33 g

Sodium citrate, dihydrate ---------------- 1.76 g (solution becomes cloudy when sodium citrate is added and stir for 30 minutes)

1N NaOH ----------------------------------- 8 ml (solution becomes clear when NaOH is added)

Distilled water ----------------------------- 30 ml

Stir for another 10 minutes and add additional 15 ml of distilled water. Store solution for 3-6 months at 4 ºC. Note: the mount of NaOH is very important. The solution should have pH 12.

EMBed 812:

Final Volume	EMBed-812	DDSA	NMA	DMP-30 (2%)
20.91 ml	10 ml	4.5 ml	6 ml	0.41 ml
31.37 ml	15 ml	6.75 ml	9 ml	0.62 ml
41.82 ml	20 ml	9 ml	12 ml	0.82 ml
52.28 ml	25 ml	11.25 ml	15 ml	1.03 ml

        Mix all four components in plastic beaker and stir with wood stick. 

Notes:

1. Shaking specimen from time to time will ensure full penetration and infiltration of chemicals.
2. Increasing the processing time for specimens with high connective tissue content such as skin, tendon, cartilage, cornea, etc. is often beneficial, especially for the 1:1 EMBed 812 and propylene oxide mixture step. Failure to increase time may result in a poorly infiltrated block and therefore weak sections which will fail under the electron beam (small holes in specimen), or even be impossible to cut in the first place.
3. Dehydration must start with low concentration alcohol (50% or 30%) and gradually increase concentration. Do not put specimen directly in 95% alcohol as this will cause incomplete infiltration.
4. To avoid moisture into specimen, use fresh opened 100% alcohol each time and change 3 times to ensure the specimen is fully dehydrated.
5. Check embedding medium expiration date. Make sure all parts of embedding medium are not more than 2 years old.