I want to investigate the differentiation between leaves of the same species that grow under different light intensities.
I think the first step is to make careful hand sections. With practice you can get excellent sections of living material. Put a piece of leaf on a slide in a drop of water and then lay another slide across the first at 90 degrees. Hold down the upper slide so that it does not move. Using a sharp razor-blade (I like the "Pal" single-edge carbon steel blades) make a single cut holding the blade against the edge of the upper slide, then a second cut with the blade angled in slightly. With practice you can get sections almost as good as paraffin sections. After getting a good look at your material that way then you can decide which embedment to use.
I think the first step is to make careful hand sections. With practice you can get excellent sections of living material. Put a piece of leaf on a slide in a drop of water and then lay another slide across the first at 90 degrees. Hold down the upper slide so that it does not move. Using a sharp razor-blade (I like the "Pal" single-edge carbon steel blades) make a single cut holding the blade against the edge of the upper slide, then a second cut with the blade angled in slightly. With practice you can get sections almost as good as paraffin sections. After getting a good look at your material that way then you can decide which embedment to use.
The method proposed above by Philip Lintilhac is very simple, quick and produces very good results. For thinner sections you have to follow paraffin sectioning or even better semi-thin sectioning using an ultramicrotime after fixation, post-fixation, dehydration and resin embedding, a procedure used for electron microscopy. This provides the best results with sections of uniform thickness of 0.5-1 μM, but it requires the use of an organized lab, experience and is time-consuming.
Dear Elaine Nunes,
It seems that you are familiar with electron microscopy judging from your picture sitting in front of a SEM(?). However, preparation for TEM is quite different, even for different plants there are differences. I attach a fixation protocol used in Prof. Peter Hepler's lab in Amherst, USA, and also elsewhere.
TEM FIXATION PROTOCOL
for Arabidopsis thaliana roots
FIXATION (in plates)
2% GA (glutaraldehyde)
5mM CaCl2.2H2O
50mM PIPES buffer
pH 7.0-7.1
2hr
RT (room temperature)
RINSE: 3X15min in rinse buffer (in plates)
TRANSFER TO LOOPS
- Root tips are transferred to loops covered with formvar film. They are stabilized in position by covering again with formvar.
- Put loops in plastic or glass vials containing 1ml rinse buffer. Mark vials clearly.
POST-FIXATION (in vials, rotation)
1% OsO4
5mM CaCl2.2H2O
50mM PIPES buffer
pH 7.0-7.1
2hr
RT
RINSE: 3X15min in rinse buffer, overnight in rinse buffer
IN BLOCK STAINING (optional) (rotation)
2% uranyl acetate in H2O
2hr
RT
RINSE: 3X15min in H2O, overnight in H2O (necessary only if block staining is done)
DEHYDRATION (rotation) (RT)
30% Acetone 30 min
50% " 30 min
70% " 45 min
90% " 1 hr
100% " 1 hr (acetone dried with molecular sieves)
100% " 1 hr "
100% " 1 hr "
INFILTRATION - EMBEDDING (rotation)
Epon/Araldite : Acetone = 1:3, 1 hr
Epon/Araldite : Acetone = 1:1, 2 hr
Epon/Araldite : Acetone = 3:1, 2 hr
Epon/Araldite pure, overnight
Epon/Araldite (next day), 4hr
Epon/Araldite, 4hr (under vacuum) (optional)
FLAT EMBEDDING
Remove root tips from loops under a stereoscope using a sharp blade and forceps. Place the root tips on fresh resin drops (Epon/Araldite) on microscope slides cleaned before at least twice with Release Agent Dry Lubricant. Mark slides clearly.
POLYMERIZATION
In oven, 70oC for 60 hr.
BLOCK MOUNTING
Detach slides and cut polymerized resin containing each one root tip separately.
Transfer roots on top of resin blocks and stabilize them in position with glue. Put them for a few hours in oven to harden.
September 6, 2014 E.P. Eleftheriou
Philip Lintilhac says the right. A hand section is better and by practice , a better section can also be obtained. You may also use piece of a potato slice. keeping the leaf section within the slice of potato, use the sharp edge of a new shaving blade. But you are to be careful to save your finger not to cut.
Firstly take a slice of a potato, cut the slice in to two parts not fully, last end will be united. place the leaf section in between the slices and hold with the thumb and index finger. keep your middle finger down the potato slice. add drop of water with the blade and drive the blade parallel to the surface of the index finger. keep the thumb down the plane of the index finger to avoid cutting. take the section in a wash glass and choose your better one.
I really Thank all of Philip Lintilhac,Farrokh Ghahremaninejad, Eleftherios Eleftheriou and Partha Pratim Dhar for your helping.
Your subject is very interesting. I"m working the similar background, but you must know each organ of cell colored with a special chemical material. For example, toluidine blue 0.5% and Borax 1% is suitable for coloring cell walls or even cuticle. Slices can do by a sharp blade, by hand or a micro tome.
I have fronted a problem at chlorophyll concentration counting and coloring. My means is which technique are used for chlorophyll coloring/staining in leaf slicing?
I need it immediately.
Thanks.
- Badges
- Science topic
- Similar topics
- Biological Science
- Botany
- Plant Biology