213 Bot Alanoud Alfagham, Iram Siddique

Bot Alanoud Alfagham, Iram siddique 213

Histologic plant tissue preparation technique

Trimming 7- Sectioning 8- Staining -6

Trimming -6 Depending on the types of embedding molds used, the wax block can be removed from the mold and mounted directly onto a suitable mounting chuck. If a paper boat method is used with a number of tissue pieces in it, the tissues need to be separated by cutting using a hot knife/spatula. The temperature of the knife minimizes the forces needed to cut the wax block, preventing uncontrolled “cracking” of the block. Traditionally, simple wood cubes can be prepared to suit a particular microtome for mounting purposes. The newly made wood cubes need to be “cured” by submerging it in molten wax for a few days before use in order to create a .better bonding between it and the specimen

Meterial

. Paraffin wax, hot plate, forceps, beckers, paper boat, ice

Protocol of mounting of Paraffin Blocks

A properly trimmed wax block needs to be mounted onto a .1 microtome chuck in order that it can be sectioned. We make use of the plastic embedding ring as our embedding chuck by filling their cavity with melted wax (this needs to be done Paraffin and Polyester Waxes at least a day in advance). Adhesive tape can be used to seal one end of the rings while wax is being poured. Once solidified, the wax in the embedding ring will provide a flat .support for the wax block

1 Bot Alanoud Alfagham, Iram siddique 213

Using an alcohol burner or Bunsen burner, heat a spatula which .2 will be used to reduce the height of the wax block and flatten its surface opposite to the samples by melting away excess wax. This procedure is preferably done in a fume hood to avoid inhaling smoke from melting wax and carried out on a sheet of aluminum foil for easy clean up. If samples are embedded in a large paper boat, the wax block will need to cut into smaller piece using a hot .spatula

Remove the plastic rings with wax. Warm both sides of the spatula .3 and place one side on top of the wax support of the embedding ring. Quickly place the sample-containing block of wax on the other side. As soon as the wax on either side starts melting, slide the spatula away and allow the two surfaces to melt together by gently pressing the top of the wax block. Release the pressure after a few seconds. Reheat the spatula and melt the edges together one more time ensuring the block is secured to the plastic microtome .chuck

Store the blocks in the fridge to ensure all the wax solidifies. .4 Depending on the procedure, the blocks can be stored in the .refrigerator or at room temperature for several months

Sectioning-7

:There are several types for Microtome such as

Rotary microtome: it use sharp knife cutting samples such as  (leaves and stems embedding in wax.(fig A

Sledge microtome: Hard materials like wood, bone and leather  (require a sledge microtome.(Fig.B

Cryomicrotome: water-rich tissues are hardened by freezing and  cut frozen; sections are stained and examined with a light (microscope. .(Fig.C

Ultramicrotome:Tissues/material are embedded in epoxy resin, a  microtome equipped with a glass or diamond knife is used to cut (very thin sections (typically 60 to 100 nanometers). .(Fig.D

Vibratome. It uses vibrating blade for cutting the samples,  sections are cut to with less pressure than required by the other (microtomes. .(Fig.E

Leaser microtome the device operates using a cutting action of an  (infra-red laser. .(Fig.F

(Fig.A). (Fig.B).

(Fig.D). (Fig.C).

(Fig.F). (Fig.E). .Method of sectioning with rotary microtome

Materials

.Rotary microtome, water bath, slides, brushes and slide warmers

Techniques for Improving the Adhesion of Cells and Tissues

Egg Albumin Adhesive

The egg albumin adhesive is suitable for attaching relatively large specimens. For preparing egg albumin coating or Mayers Albumen solution, mix thoroughly 50 mL of fresh egg white and 50 mL of glycerol, using a magnetic stirrer. Add 1 mg of sodium azide as a preservative.The mixture can be stored for several weeks at 4 °C. Apply a small drop of adhesive coating on a clean slide or cover slip and spread evenly with a clean finger until a thin film remains. The coated slides or cover slips are stored in a dust-free box at room temperature and must be used in a few days. Long storage is not suitable because the adhesive .strength will decrease due to drying

Protocol

Using a single-edged razor blade trim the block to the appropriate .1 size and ensure that the top and bottom edges are parallel to one another and the trims are clean. This ensures the formation of a .“straight” ribbon

Depending on the size of the block face, the cutting angle should .2 be set at 5–10 while the section thickness can be set between 5 and 10 μM. Generally speaking, the smaller the block face, the smaller .the angle, and the thinner the sections that can be obtained

During sectioning the first sections can be discarded if they do not .3 .contain any sample. With practice, a ribbon of sections will form

A small wet brush is used to stick and hold the ribbon at one end, .4 gently lift it up from the knife without detaching it from the knife- .edge

Continue to section. Once the desired length is reached, using .5 another wet brush gently lift the entire ribbon and place it water bath 37c . The ribbon will expand on the warm water removing the .compression marks generated during sectioning

Applay the Mayers albumen on slides before applay ribbons into .6 .slides to will adhere between ribbons and slide

.Pick up the ribbons from the water bath into the slides .7

Depending on the size of the ribbon, if there is space for a second .8 .row, an additional ribbon can be placed next to it

Be sure to place the shiny side down onto the slide as it is smooth .9 .and will adhere to the slide surface better when dry

place the slides onto another slide warmer set at 30 °C overnight. .10 Just before drying, if necessary, the sections can be examined using a microscope. Once fully dry, the slides can be stored in a slide box (.and used in further procedures.(Fig G

Defects

There are a number problems associated with paraffin wax sectioning. ;The most common problem are

the generation of static electricity during sectioning, especially in a  .dry environment

Shattering 

Stripes on the section 

"Wavy lines "Chatter 

.(Fig(G

Staining-8 After fixation, embedding, and sectioning, structural details within cells and tissues are difficult to be resolved. In order to examine the structural organization, sections need to be stained. The purpose of staining is simply to increase the visibility of biological specimens. A majority of plant tissues have little color; hence, little contrast is present to show surface and internal features. One way to increase the contrast of a specimen is through staining. Furthermore, proper selection of staining methods can provide specific histochemical information and define .cellular components clearly

To staind the tissue it should pass through several gradual alchol :consentration and days please follow the table below

Time Chemicals min 20 Xylene absolute min 1-2 Xylene: Ethanol 1:1 min 1 Ethanol 100% min 1 Ethanol 96% min 1 Ethanol 80% min 1 Ethanol 70% min 1 Ethanol 50% min 30 Safranine day min 1 Ethanol 50% min 1 Ethanol 96% min 1-2 Light green day min 1 Ethanol 100% min 1 Ethanol 96% min 1-2 Xylene : Ethanol 1:1 min 1 Xylene absolute

Summary of Microtome protocol

Trim the paraffin containing the object into a convenient shape,  .and fasten it upon a block of wood

The tissue is then cut in the microtome at thicknesses varying from  .2 to 25 micrometers thick

.Place paraffin ribbon in water bath at about 40-45 ºC 

.Apply Mayres albumen onto slides 

Mount sections onto 

.slides 

Allow sections to air dry for 30 minutes and then bake in 45-50 ºC  .oven overnight

Pass the slides in different gradual alchol and days to stained the  .tissues

.Apply candapalsam onto the slides allow slides to air dry for 1 min 

From there the tissue can be mounted on a microscope slide,  .examined using a light microscope

NEVER allow baking temperature go higher than 50 ºC for  sections thicker than 25um. Otherwise sections may crack, especially 25-50 um thick sections, and result in sections falling off .slides during staining