1.
Clear a space for the operation
and lay out all the instruments that will be needed.
2.
Weigh the rat and determine the
dosage of xylazine and ketamine
at a dose of 10 mg/kg xylazine
and 100 mg/kg ketamine.
It
is e
For
all three methods described, a 1 ml syringe is used with a 25 Gauge needle (0.5
mm external diameter) of 1.5 cm length. A relatively small and short needle is
nece
The
cloth is folded over the head of the rat and the body, exposing the tail. Now
pre
The
thumb is placed on the caudal side of the tail base and the rest of the hand is
placed cranially from the tail, which fixes the rat firmly and gently. The hind
part of the body is now lifted carefully, until the abdomen becomes visible. If
not one bends the rat´s back carefully, it may give damage, pain and
resistance, which is to be avoided.
The
intraperitoneal injection can now be performed with
the right hand. The injection must be given lateral from the linea alba at a horizontal level
between the knees. After one has given the injection, the rat can be put back
into the cage. One has to be careful that one does not damage the toe nails
while doing this: often the rats have grasped onto the cloth, so one has to
remove the cloth carefully from the rat.
If the animal is too lightly anesthetized, he
will respond by struggling if his tail is firmly squeezed with a pair of
forceps. Similarly, he will withdraw his
rear paw if it is firmly squeezed with a pair of forceps. Another method of
testing for the depth of anesthesia is to squeeze the animals
ear. If he is not properly anesthetized,
he will respond by moving his paw.
One of the last reflexes to disappear in an
animal as the anesthetic takes effect is the eye-blink reflex. Gently stroking
of the animals eye with a saline-moistened cotton-tipped applicator will
determine whether the eye-blink reflex is present or whether the animal has
been succe
Recommended
Volume
from Stock
Rat Weight (gms) Ketamine (mg) Xylazine
(mg) Ketamine
50 ug/ml Xylazine 20 mg/ml
|
150 |
11.3
- 15.0 |
1.1
- 1.5 |
226
-300 |
55
- 75 |
|
175 |
13.1
- 17.5 |
1.3
- 1.8 |
260
- 360 |
65
- 90 |
|
200 |
15.0
- 20.0 |
1.5
- 2.0 |
300
- 400 |
75
- 100 |
|
225 |
16.9
- 22.5 |
1.7
- 2.3 |
340
- 460 |
85
- 115 |
|
250 |
18.8
- 25.0 |
1.9
- 2.5 |
380
- 500 |
95
- 125 |
|
275 |
20.6
- 27.5 |
2.1
- 2.8 |
420
- 560 |
105
- 140 |
|
300 |
22.5
- 30.0 |
2.3
- 3.0 |
460
- 600 |
115
- 150 |
|
325 |
24.4
- 32.5 |
2.4
- 3.3 |
480
- 660 |
120
- 165 |
Materials:
1.
Large live rat, balance for
weighing rat
2.
Scalpels with # 10 and #11 blades
3.
Large and fine forceps
4.
Good quality fine and Mayo di
5.
Gelfoam
sponges
6.
Probes
7.
Latex gloves
8.
Cotton and gauze pads, Q-tips
9.
Xylazine
20 mg/ml stock, Ketamine 50 mg/ml stock. Normally
make up a 1:4 mixture of xylazine to ketamine for
injections. This gives a working solution that reflects the 10 mg/kg xylazine and 100
mg/kg of ketamine that is recommended. Next
calculate the volume of each drug that is required,
add the volumes together and use that volume of the mixture for injection.
10. Tuberculum syringes with short needles
A. Fixation
A good fixative should kill and
preserve tissues without any major alterations in their structure. It should
stabilize carbohydrates, lipids, proteins and nucleic acids to prevent them
from being dissolved or redistributed during subsequent processing. There is no
ideal fixative suitable for all tissues. The most common are formaldehyde, glutaraldehyde, osmium tetroxide,
mercuric chloride, potassium dichromate, picric acid, ethanol and acetic acid.
A common fixative for routine histology
is Neutral Buffered Formalin. We will use this fixative in our preparations. It
preserves the basic tissue types (epithelium, connective, muscle and nervous)
so they can be easily recognized. One flaw is that it does not stabilize
lipids. Organelles such as mitochondria, Golgi bodies and endoplasmic reticulum
contain a high percentage of lipid and are rendered
unrecognizable. Bouin's fluid coagulates proteins.
Delicate filaments and microtubules are totally disorganized. Although the nucleus and chromosomes are generally rendered easily
stainable.
In our procedure small pieces of tissue
(5-10 mm3) are usually fixed for overnight at room temperature.
Larger pieces may not be properly fixed at the center. Following the fixation
the specimens must be washed to remove the fixative this is usually done with
water or ethanol. We will use 70% ethanol. The washing will be accomplished by
placing the specimen into two changes of 70% ethanol. Your specimen can be kept
for several months in ethanol, although the sooner it is embedded the better.
You will prepare 3 samples of your tissue.
B. Dehydration
The next step in the process involves the
removal of water from the specimen. This is accomplished by placing the tissues
in a grades series of ethanol or other dehydrating agents. This is a necessary
step for subsequent paraffin infiltration. You should plan your time wisely at
the onset of this step because it commits you to carry your specimen through
the process of embedding.
There are trade-offs in dehydration as
in all the other steps. We want the tissue to remain in each of the
concentrations of ethanol long enough for the water to be removed, but exposure
of the tissue to 95% or 100% ethanol for too long a period of time hardens to
tissue excessively and sectioning is difficult. Also care must be taken not to
let the tissue dry out while moving it from one solution to the next.
C. Clearing
The dehydrating agent is replaced with
a substance called an antemedium. This is a general
name for a substance that will mix with both the dehydrating agent and the
embedding medium. Toluene or xylene are commonly used antemedia.
A property of these materials is that
they have a high refractive index and tend to clear the tissue. They are
therefore often called clearing agents. If you have properly dehydrated your
tissue sample, when placed in the clearing agent it should appear clear and
translucent. If it appears milky it has not been dehydrated properly.
There are problems with clearing
agents. They are highly volatile and if the tissue is exposed to the air the
clearing agents will evaporate and leave air pockets in the tissue. Another
concern that is shared with ethanol is these compounds are highly flammable and
appropriate care must be taken with their use.
D. Infiltration and Embedding
Cleared specimens are placed in a
solution of molten paraffin in an oven set at about 60o C. The antemedium is gradually removed by dilution in several
changes of paraffin. The paraffin infiltrates the tissue and more or less takes
the place of water in the living tissue. This procedure causes the tissue to
shrink down to as much as 40-50% of its original volume. Maintaining the tissue
in the oven may also cause the tissues to become hard,
especially is the temperature is too high, and as we will see hard blocks are
hard to section.
The tissues are finally transferred to
a small container of pure molten paraffin and oriented in the direction they
will be sectioned. The paraffin containing the specimen is rapidly cooled and
solidified by placing it in ice water. The protocol we will use is as follows
(3 blocks):
1. Neutral
Buffered formalin 2 hours to overnight
2. 50% ETOH 2
hours
3. 75% ETOH 2
hours
4. 95% ETOH 2
hours
5. 100% ETOH 1
hour
6. 100% ETOH 1
hour
7. 100% Organic
Solvent 1
hour
8. 100% Organic
Solvent 1
hour
9. 100% Molten
Paraffin* 2
hours
10. 100% Molten
Paraffin* 3
hours to overnight
11. Embed and
orient tissue in block
12. Place in ice water to solidify * The Best Kind of
Paraffin is: Baxter Scientific Products Ameraffin
Tissue Embedding Medium
Cat. # M7346-1A
E. Sectioning
The tissue sample now surrounded by
hard paraffin is trimmed to a trapezoid shape and placed in the chuck of a
microtome. A microtome is a instrument for holding and
advancing the specimen while moving it past the cutting edge of a sharp knife.
Paraffin specimens are usually cut at thicknesses of 5-10 microns. Sections are
cut then removed from the blade with a small paint brush and placed on a slide
covered with a thin layer of albumin and water (4 drops of water).
The albumin acts as an adhesive and the
water allows the section to expand before it attaches itself to the slide. The
slides are then placed on slide warming trays to help flatten the sections out
(1 hour+). Sectioning is the most difficult part of the tissue preparation
process and you should read chapters 4 and 5 in Humason
(1979) before signing up to do your sectioning. Your initial sectioning
attempts should be done in the presence of the instructor or lab assistant.
Everyone encounters difficulties in the process of sectioning,
here are some suggested remedies (Modified from Richards, 1949).
1. Ribbons are crooked
1.1 Wedge-shaped
sections caused by poor trimming; sides of paraffin block are not parallel or
not parallel to edge of knife.
1.2 Part
of knife edge may be dull; try another part of it.
1.3 Uneven
hardness of the paraffin; one side may be softer than the other, or contain
areas of crystallization; re-embed.
2. Sections fail to form ribbons (usually
due to hardness of paraffin)
2.1 Use
softer paraffin (lower melting point).
2.2 Blow
on knife to warm it or dip it in warm water.
2.3 Cut
thinner sections.
2.4 Place
table lamp near knife and block to warm them both.
2.5 Resharpen knife.
2.6 Lessen
tilt of knife and clean edge.
2.7 Dip
block in softer paraffin and retrim so a layer of
this paraffin surrounds original block.
3. Sections are wrinkled or compressed
3.1 Resharpen knife; a dull knife compresses badly.
3.2 Paraffin
too soft; re-embed in harder paraffin.
3.3 Cool
block and knife.
3.4 Increase
tilt of knife.
3.5 Clean
edge of knife with finger or xylene; remove any
paraffin collected there.
3.6 Tissue
is not completely infiltrated (Poor infiltration is usually caused by traces of
water or alcohol). Correct by first removing paraffin that is present. Soak in xylene for
2 or 3 hours (or more); change twice, then place in absolute alcohol for 1 or
more hours. This should remove all
traces of water. Clear again in xylene (check against milky appearance); reinfiltrate and embed.
3.7 Soak
tissue block in water. When soaking in
water is recommended, the cut face of the tissue is exposed to tap water for 30
to 60 minutes. This treatment is
generally satisfactory; however, some technicians advocate the addition of
glycerin (1 part to 9 parts water) or fabric softeners, same ratio, or several
drops of liquid dish detergent per 100 ml of water, or 60% alcohol instead of
water (Lendrum 1944).
These fluids work in through the cut tissue surface and soften tough
parts. (Exception: Do not soak nervous system tissues at any time and lymph
nodes and fatty tissue only briefly. Paraplast will not absorb water.)
4. Ribbons are split or scratched
longitudinally
4.1 Nick
in knife; move to another part of edge or resharpen
knife.
4.2 Knife
dirty or gritty along edge.
4.3 Dirt
or hard particles in tissue or in paraffin; crystals from fixing solution not
adequately removed; filter stock paraffin or decalcify tissue.
4.4 Decrease
tilt of knife.
4.5 Tissue too hard; soak in water or RDO decalcifier
briefly (1 to 5 minutes).
5. Tissue crumbles or falls out of paraffin
5.1 Poor
infiltration; reinfiltrate and re-embed.
5.2 Not
completely dehydrated.
5.3 Not
completely de-alcoholized.
5.4 Too
long in paraffin bath or too hot while there; soak in water.
5.5 Clearing
fluid made tissue too brittle; soak in water.
6. Sections cling to block instead of knife
6.1 Knife
dull or dirty.
6.2 Increase
tilt of knife.
6.3 Paraffin
too soft or room too warm; try harder paraffin or cool block.
6.4 Infiltrating
paraffin too hot, or too long exposure to solutions that harden; soak in water.
7. Tissue makes scratching noise while
sectioning
7.1 Tissue
too hard; paraffin too hot or exposed too long to solutions that harden; soak
in water.
7.2
8. Knife rings as it passes over tissue
8.1 Knife
tilted too much or too little.
8.2 Tissue too hard; soak in water.
8.3 Knife
blade too thin; try a heavier one.
9. Sections curl, fly about, or stick to
things, owing to static electricity from friction during cutting,
especially in weather of low humidity.
9.1 Increase
humidity in room by boiling water in open pan.
9.2 Ground
microtome to water pipe.
9.3 Postpone
sectioning until weather is more humid; early morning sectioning often is best.
9.4 See
p. 60 for suggestions concerning clothing, furniture, and static eliminator.
10. Sections are skipped or vary in thickness
10.1 Microtome
in need of adjustment or new parts.
10.2 Tighten
all parts, including knife holder and object holder clamp. Always!
10.3 Knife
tilt too great or too little.
F. Staining and Mounting
The last step of the process involves
the removal of the paraffin from the section, the hydration of the section and
finally the staining of the thin section of tissue. There many different stains
and staining techniques. We will use one of the common staining procedures, hematoxylin and eosin or the H and E staining procedure.
Hematoxylin stains the nuclear materials and the
eosin stains the cytoplasmic elements of the cells.
During this portion of the process you should wear old clothes or lab coats.
You should also understand the whole procedure before you get start. There are
a number of different protocols for staining the one we will use is as follows:
1. Place slide in
solvent for 10 minutes (removes paraffin)
2. Place slide in a
second change of solvent for 10 minutes
3. Place in 100% ETOH
for 3 minutes with agitation (hydration)
4. Place in second 100%
ETOH - up and down motion 10X (hydration)
5. Place in 95% ETOH
for 3 minutes, agitate gently (hydration)
6. Place in 80% ETOH
for 3 minutes, agitate gently (hydration)
7. Place in distilled
water for 3-5 minutes, agitate gently
8. Place in Harris Hematoxylin stain for 3-5 minutes
9. Rinse in lightly
running water
10. Examine wet slide
under microscope, if too proceed to step 11, if too light return to step 6, if
just right proceed to step 12
11. Remove excess stain
with acid-alcohol (35% ethanol with 0.1 m HCl)
12. Neutralize in
alkaline alcohol (35% ethanol with pinch of NaCO3)
13. Place in 70% ETOH
for 1 minute (dehydration)
14. Place in eosin in
70% ETOH for 2-5 minutes (counterstain)
15. Place briefly in 70%
ETOH to rinse off excess eosin
16. Place in 95% ETOH
for one minute (dehydration)
17. Place in a second
change of 95% ethanol for 1 minute (dehydration)
18. Place in 100%
ethanol for 1 minute (dehydration)
19. Place in 100%
solvent for 2-10 minutes (clearing)
20. Add small drop of
mounting media and a cover slip
21. Allow to dry before
examining
References
1. Galigher, A. E. and E. N. Kozolff.
1971. Essentials of practical microtechnique. Lea and Febiger, Philadelphia.
2. Humason, G. 1972. Animal Tissue
Techniques. Fourth Ed. W. H. Freeman & Co. San
Francisco.
3. Sheehan,
D. C. and B. B. Hrapchak. 1980. Theory and Practice
of histotechnology. The C. V. Mosby Co. ,
4. Clark,
G. 1981. Staining Procedures. Fourth
Edition. Williams & Wilkins.
IV. PARTS
OF A TYPICAL CELL
A good example of a typical cell is a
lymphocyte, one of the white blood cells. Lymphocytes can be seen in slides of
blood smears (slide 25). Find this slide in your set and examine under low
power. Identify the numerous erythrocytes (red blood cells) which appear pink
and are non-nucleated in mammals. Generally one or two lymphocytes appear in
each low power view.
Lymphocytes can be identified by their
large nucleus with masses of chromatin material. The cytoplasm is a clear
region that is so small compared with the nucleus that it often appears as a
small rim at on side of the cell. Examine the
lymphocytes at all magnifications. Draw the cell as it appears under high
power.
This is a good opportunity to be able
to examine a typical cell at higher magnification and resolution using electron
micrographs (photographs of cells taken on a transmission electron microscope).
Micrographs such as these are used extensively today and you will be seeing a
lot of them both in your text and in the lab. Within a short time you should be
able to recognize the greater resolution that characterizes an electron
micrograph from a light micrograph (photograph from a light microscope).
Examine the electron micrographs in chapter 1 of the text and in addition to
the structures listed above, make drawings and be able to identify the
following structures:
1. Ribosomes (15 nm)
2. Endoplasmic reticulum (both
smooth and rough)
3. Golgi apparatus
4. Mitochondria (0.5 um)
5. Lysosomes
6. Microvilli
7. Filaments
8. Inclusions
9. Cell membrane and cell division
10. Nucleus and nuclear membrane