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(1) Tissue that has not been fixed - go
immediately to glutaraldehyde fixative. Tissue must be minced
very fine (no more than 1mm) on either a piece of dental wax or
parafilm.
(2) Tissue fixed in Sorensen's formalin
should be washed overnight at 0-4°C in PO4 buffer
(Ph. 7.4) buffer.
(1) Cells can be fixed either as a monolayer
or in a suspension depending on how you want to study the cells.
(1) Animals fixed in situ must be
properly anesthesized and heparinized. Procaine and Ringer's
solution should be administered prior to the fixation process.
1) Glutaraldehyde 3% - 3hrs. 0-4C°
2) Wash PO4 buffer - overnight 0-4C°
3) OsO4 1.0% - 1 hour
4) Wash PO4 buffer - 1 hour
5) 30% acetone 10'
6) 60% acetone 10'
7) 90% acetone 10'
8) 100% acetone 5' 3x (this acetone is made anhydrous by placing
it in anhydrous sodium sulfate)
9) 1 part LX112-Araldite / 2 parts Acetone
– 30’
10) 1 part LX112-Araldite / 1 part Acetone -
30'
11) 2 parts LX112-Araldite / 1 part Acetone
- 30'
12) Pure LX112-Araldite - 30'
13) Embed in a BEEM capsule, gelatin
capsule, or flat embedding mold. If a BEEM capsule is to be used
fill the capsule only 3/4 of the way
LX112- 50ml
(25ml;12.5ml)
DDSA- 110ml
(55ml;27.5ml)
ARALDITE 6005- 40ml
(20ml;10ml)
DIBUTYL PTHALATE
2ml (1ml;0.5ml) (determines the
hardness of the blocks)
Mix the above plastics using a mechanical
mixer until a uniform color is achieved - then add 4.6ml
(2.3ml;1.7ml) of BDMA (this is the polymerizer)
PROCEDURE FOR PROCESSING CELLS IN A PELLET
1- Fix suspended cells 30-60 minutes in
glutaraldehyde (1.5% or 3.0%). (Pellet should become suspended
with addition of fixative)
2- Spin down cells and carefully pour off fixative. Do not use a glass pipette.
3- Resuspend cells in Phosphate buffer and wash for one hour.
4- Post-fix cells in osmium tetroxide for 30 minutes.
5- Wash in buffer and dehydrate and process as described above except that all steps have to be followed by centrifugation.
6- At the last pure epon-araldite step
approximately 1ml of epon is added to the centrifuge tube and
suspended with a wooden applicator stick. Carefully pour
the plastic into labelled conical or parabolic BEEM capsule.
7. Place these BEEM capsules into a plastic centrifuge tube and spin down for at least one hour to concentrate the sample.
8. Polymerize as usual except that after
polymerization the centrifuge tube may have to be broken to
remove the BEEM capsule if any plastic was deposited between the
BEEM capsule and the wall of the centrifuge tube.
If BEEM capsules are to be used labels are written in india ink with a crow quill pen on white paper. Labels are cut and placed within the BEEM capsule
PREPARATION OF SOUTIONS
1- Sorensen’s PO4 Buffer pH 7.4 --------- Stock Solution
A - 0.2 M monobasic sodium phosphate (27.8 gm/ 1000 ml .H2O)
B - 0.2 M dibasic sodium phosphate (Na2HPO4 -7H2O) (53.65gm/1000 ml D H2O)
Take 81 ml. of solution B and set in pH meter. Add solution A until the pH is 7.4 (add approximately 19ml of A). Then add 100 ml. D.H2O (to regulate molarity). Total volume should be approx. 200ml
2. 3.0% glutaraldehyde in
PO4 Buffer (pH 7.4)
A - Take 6 ml of stock glutaraldehyde (50%). Put in volumetric flask and QS to 100ml. Add 0.8 ml of 1.0% CaCl2 and shake.
B - Take one 5 ml vial of 70% glutaraldehyde add 116.6 ml of PO4 buffer and to this add .97 ml of 1.0% CaCl2 solution and shake. ( N.B. This method of fixative preparation uses a highly purified glutaraldehyde especially prepared for electron microscopy.)
3. 1.0% Osmic Acid
(pH 7.4)
Dissolve ½ gm osmium tetroxide in 50
ml PO4 buffer.
LR
WHITE - ELECTRON MICROSCOPIC IMMUNOCYTOCHEMISTRY
50%
purified glutarldehyde 2.0ml
2ml saturated
aqueous picric acid - 15ml
0.1M sorensen's phosphate buffer pH 7.3 - 83ml
70% ETOH 30 minutes 2x
2 parts LR white to 1 part 70% ETOH - 1hr
pure LR white -
1hr
pure LR white -
overnight on a rotary shaker
embed in LR
White
cure in a vacuum oven at 50øC. for 24 hrs. (temperature of the oven should never go over 55C°.)
Section the
material and do the immmunolocalization technique.
You can
counterstain with lead citrate for 3 minutes. Routinely sections
should be stained with uranyl and lead to check out themorphology
of the tissue.
A - Prepare 1.0% Methylene Blue in 1.0% aqueous Sodium Borate
B - Prepare 1.0% aqueous Azure B.
1 - Place 0.5u to 1.0u sections in a small drop of water on a CLEAN glass slide.
2 - Heat GENTLY over an alcohol lamp flame and place slide on a table until water evaporates. Make sure that the slide and sections are dry or they will lift off when you add stain. Heating the slide helps stretch out wrinkles that have been produced in the section and as the water eva porates the section becomes firmly attached to the slide and can be stained. The heat must not be too intense or air bubbles will be formed under the section and may become trapped as the section dries.
3 - Stain slide with a one to one mixture of solution A and B for 30 seconds or as long as 30 minutes or until stain is about to dry around its edge. Gentle heat sometimes help. If the sections stain very lightly the section was probably cut too thin or the stain is worn out.
N.B. - The staining solution should always be freshly filtered before use. The stock staining solution can be kept indefinitely but the mixture of stains should be prepared fresh each day
4 - Wash excess stain from the slide and sections with DISTILLED water.
5 - Dry bottom of slide and place on a slide warmer for AT LEAST 1HR. If this step is hurried water will not be totally evaporated and will cause cloudiness when mounted with permount.
6 - Mount slide with a coverslip using permount.
This method can be used either on small blocks of tissue (1mmx1mm) or cells or organisms grown on cover slips. Be very careful handling the specimens in order to avoid artifact.
1) Glutaraldehyde 3.0 % - 3hrs. 0-4°C
2) Wash PO4 buffer - overnight 0-4°C
3) 30% acetone 10'
4) 60% acetone 10'
5) 90% acetone 10'
6) 100% acetone 5' 3x (this acetone is made anhydrous by placing it in anhydrous sodium sulfate)
The specimens are placed in the Critical Point Dryer (CPD)) and dried. Technique will be demonstrated.
Specimens are then placed on sticky cleaned stubs, attached with some silver paint and either metal (platinum) or carbon coated.
1 - Remove plastic cover.
2 - Center glass using a ruler on the top plate against the back stop plate. Support ends of glass rectangle if the rectangle is very long otherwise excessive pressure will be placed on the score line.
3 - Check glass to ensure that the manufacturer's score is down (This side is identified by minute ridges along the edge)
4 - Turn the handwheel clockwise while holding the glass in place. After the bumpers have made contact with the glass, complete on more full turn of the handwheel.
The glass should be held securely. Try to rock the anvil to see if the glass is secure. If the anvil can be moved, continue turning the hand wheel RAPIDLY until the anvil is tight.
5 - Pull the plunger adjustment shaft out to prepare for the short stroke.
6 - Pull the scoring shaft all the way out. Depress the button and push the scoring shaft back with one even stroke to score the glass.
7 - SLOWLY, GENTLY, and CAREFULLY turn the breaking control knob clockwise until the glass breaks.
8 - Turn the handwheel counterclockwise to raise the clamp and anvil
9 - Remove glass by placing your fingers on top of the glass and sliding the pieces toward you.
Repeat all steps until glass is broken into 2.54 cm squares.
_______________________________________________________
1 - Inspect the corners and sides of the square carefully. Select the corner that is the smoothest ( with the fewest score marks) and the most nearly perpendicular to the 2,54cm square.
2 - Place the glass square on the top plate with the selected corner seated in the back stop plate registration notch. The scored edges of the glass square should now be up
3 - Push the positioner slide lightly against the corner of the glass facing the operator.
4 - Hold the positioner slide lightly in place while turning the handwheel clockwise to clamp the glass to the top plate.
5 - Push the plunger adjustment shaft IN for a long stroke.
6 - Pull the positioner back so it is flush against the top plate surface.
7 - Pull the scoring shaft all the way out. Depress the button and push the scoring shaft back in one even stroke.
8 - Place plastic cover over the instrument.
9 - SLOWLY AND CAREFULLY turn the breaking control knob CLOCKWISE until the glass breaks.
10 - Remove the plastic cover.
11 - Turn the handwheel counterclockwise to raise the clamp arm and anvil.
Inspect each glass knife
The outer one-third of the knife edge that parallels the stress line will be the sharpest section.
The final test of the acceptability of the glass knife is to examine the knife edge for "whiskers". This is done under a binocular stereo microscope with bright field illumination from above against a dark background.
1. Boiled Distilled Water - 1'
2. Stain with
Uranyl acetate in a small Petri dish - 30' (occasionally shake
grids.
(N.B. To prevent evaporation place a small Petri dish in a large Petri dish filled with methanol. Cover large dish.)
(Leave grids in the uranyl acetate section side up - shinny side down)
3. AS QUICKLY AS
POSSIBLE -Transfer grids into 3 washes of absolute mentanol -
Do not let grids dry.
4. Wash in a 50% mixture of water and
methanol
5. Wash in boiled distilled water - 3x
6. Stain in a waxed Petri dish with Lead
Citrate - 5'
7. Wash in 3 changes of boiled distilled
water.
1- Dissolve 5g of uranyl acetate in 20ml of absolute methanol (solution is saturated). Date and place solution in a brown bottle in a refrigerator. Solution is stable for one month.
2- When ready to use - filter in a funnel with very fine filter paper.
To prevent evapoation cover the top of the funnel with a Petri dish. Add a few drops of absolute methanol to the filtered staining solution.
1.33 G Lead Nitrate
1.76 g Sodium Citrate
Combine to 30ml with boiled water. Shake for 2 minutes and let stand for 30 minutes. Add 8.0ml of 1N NaOH. Shake vigorously
QS to 50ml with boiled distilled water
Remove stain from center of volumetric flask.
Stable for not more than 1.5 to 2 weeks. Date and place in refrigerator.
Make sure that the x-ray dewar is filled to 90% of capacity at all times. The unit has to be filled at least twice a week.
The Z axis of must be set at 33
The dectector must be cranked into the column until it is in the proper position.
Turn on the syquest drives
Turn on the thermal printer
Turn on the monitor
Place cartridges in drives:
1. DL0 - system files
2. DL1 - data files
Press program key and enter
press 5 and enter
boot enter
If the computer is in the:
@ prompt (ODT) type 173000G
. prompt (TSX) type q for Quantex
quantex to return to TSX press Control C
For example:
resolution = # channels x10ev + H+ L
Regular f1.2 lens
ASA 12
1 sec
f8 (actually f4 to f8)
Macro lens
ASA 1
1 sec or 1.5 sec
f8 (actually f4 to f8) 5.6 works best
for one role of film - 4 oz of kodalith developer A
4 oz of kodalith developer B
developing time - 2 min 45 sec. 20C°
WASH:
running tap
water - 3 changes
FIXATION:
4 to 5 minutes
WASH
20 to 30 minutes
PROCEDURE FOR DEVELOPING ELECTRON MICROSCOPE PLATES
1.
Remove Plate from metal holder
2.
Place plates into film holders
3. Check to see that the developer (D-19; 1:1) is at 68°F If developer is too warm , cool with an ice cube in a plastic bag and if too cold use the warming iron.
4. Develop plates for a maximum of 4 minutes. You have to watch the plates to make sure they don’t under or over-develop.
5. Rinse plates in running tap water (68°F) for 1½ minutes.
6. Fix plates in fresh fixer for 10 minutes
7. Wash plates in clean running tap water (68°F) for ½ to 1 hour
8.
Dip plates in PHOTO FLO (optional)
9. Hang plates to dry
SCANNING ELECTRON MICROSCOPE
TURN ON:
1. Turn on the main power switch on the back wall of the room.
2. Turn on the water valve all the way - the water pressure on the last valve should read 10psi.
3. Throw the EVAC POWER switch in the up position. This switch is found on the front panel of the microscope under the column.
4. Press the silver/black EVAC SWITCH on the top of the microscope panel next to the column.
5. Turn on the Air conditioner in room 141b.
WAIT 20 MINUTES BEFORE USING THE MICROSCOPE
TURN OFF:
1. Press the silver/black STOP SWITCH button on top of the microscope panel next to the column. Wait until the control motor stops and the RED STOP LIGHT goes on.
2. Throw the EVAC Power switch the down position.
3. Throw the MAIN Electronic switch in the down position
WAIT 20 MINUTES
4. Turn off the water
5. Thlrow off the main power switch on the wall