Histopathologic Techniques - RNA Extraction

RNA extraction. Faults/Problems Observed during Section-Cutting Difficulties encountered during cutting of sections are mostly due to faults encountered during the processing of tissues or due to some faults in the technique or cutting itself. Therefore, it must be evaluated and corrected on a case-to​case basis, if good tissue sections are to be made. Below is a summary of the most common problems that can be identified at the microtome. Many microtomy problems have several causes so it is best to address them one at a time until the problem is resolved. Fig. 13-4. Section detached from the slide due to faulty processing PROBLEM Sections fail to form ribbons Sections roll up on cutting so that they adhere and get broken against the knife edge Ribbon is curved, REASON REMEDY Surfaces and edges of the block are not Re-trim the block parallel Horizontal surface Re-adjust and reof the block is not orient the block parallel to the knife Coat horizontal Paraffin wax is too edges of the block hard with wax of lower melting point Knife is tilted too Reduce the tilt much Readjust the Sections are too thickness of the thick sections Knife is dull Hone and strop Knife is blunt Sharpen the knife Tilt of knife is too Reduce the tilt great Knife edge is dirty Clean the knife edge Adjust the knife so Blunt or dull spot that knife edge will on the knife, present a uniformly producing an sharp edge to the irregular knife edge block, or sharpen crooked or uneven instead of straight Sections are compressed, wrinkled or jammed Edges of the block are not parallel but round or wedge shaped Knife is not parallel to the block Knife is blunt or dull Paraffin block is warm and soft Knife edge is coated with paraffin Sections are too thin Microtome set screw is loose Tilt of knife is too vertical Sections are squashed (width of each section is less than that of the block) A hole is formed in the section Sections of unequal thickness are produced Bevel of knife is lost due to incorrect sharpening Re-trim the block Readjust the knife and block Re-sharpen the knife Cool the block on ice water until firm Clean the knife edge Readjust thickness of the section Tighten the screw Reduce the tilt Re-sharpen, using a knife back or automatic knife sharpener Re-embed in freshly filtered wax if necessary Bubble or dirt formed in the embedding medium Tissue is not processed properly and will not form a Re-process tissue section (especially if center is raw) Under-processed portion of tissue Re-process tissue bursts on contact with warm water Once embedded in paraffin wax, decalcification is Hard spot in tissue impractical; use a due to calcium base-sledge microtome with a wedge knife Tilt of knife is too great or bevel is not cleared, hence Reduce the tilt object is compressed against the knife edge Clamp set screw on knife or block Tighten the screw holder is loose Cut blocks into smaller fragments Soften the blocks in Blocks are too hard detergent or phenol Breathe out or blow gently on the bock Static electricity due and knife to break up to low atmospheric static electricity, or boil water in the Sections adhere to the humidity room to increase knife or other parts of humidity the microtome Knife edge is dirty Clean the knife edge Knife edge is dull Sharpen the knife Knife tilt is too Reduce the tilt great Nicks or damage on Sharpen the knife the knife edge Re-embed in freshly Ribbon is split or Dirty embedding filtered wax lengthwise vertical scratches are seen on Clean knife edge Knife edge is dirty sections with xylene Tilt of knife is too Reduce the tilt great Knife tilt is too Reduce the tilt great Sections are lifted Knife is dull Sharpen the knife from the knife on Paraffin is too soft upstrokes Cool paraffin wax in or room temperature ice water is warm Tilt of knife is too small, paraffin Resistance is felt on block is therefore the lower part of the compressed against Increase the tilt section during cutting the base of the knife towards the end of stroke Knife edge vibrates Treat with phenol Horizontal or parallel due to hardness of during processing or lines or furrows across tissue collodionize the section Tilt of knife is too ("chatters") are seen Reduce the tilt great Knife is blunt Sharpen the knife Adjust the knife so that knife edge will Knife is not present a uniformly clamped properly sharp edge to the block, or sharpen Section cut is sometimes thin, Knife or block Tighten adjusting sometimes thick holder is loose and locking screws Blocks are too large Knife makes a hard metallic scraping or ringing sound on backstroke, when section is cut Frozen tissue crumbles and comes off the block holder when cut Frozen tissue chips into fragments when cut Ribbons are crooked Sections are too thick On trimming, tissue smells of clearing agent Tissue is opaque, section cutting is difficult due to presence of alcohol Tissue shrinks away from wax when trimmed Knife tilt is too small that block is compressed by bevel and section is not cut Tilt of knife is too slanted or too big Increase the tilt Readjust the tilt Tissue is too hard Take fresh block treated with phenol during processing Knife blade is too thin Change the knife Freezing is not adequate Refreeze the tissue block Tissue is frozen too much Warm the tissue with the fingers Top and bottom edges of block are not parallel to edge of blade/sides of block are not perpendicular to the blade Wrong micrometer setting Adjust the block holder to make the block edges parallel to the knife Microtome needs recalibration Block is trimmed down nearest to the tissue. Remaining wax is melted on Clearing agent not embedding oven and completely removed paraffin due to insufficient impregnation is impregnation repeated, changing the paraffin at least once before embedding Repeat clearing; if object has already been embedded, Insufficient clearing prolong clearing up to 12 hours, then reembed Insufficient dehydration, Repeat the whole therefore procedure incomplete clearing and impregnation On trimming, wax appears crystalline Contaminated wax Block not cooled rapidly enough Paraffin block, after cooling, is moist and crumbles Insufficient paraffin Re-embed in freshly filtered wax Re-embed in freshly filtered wax Repeat paraffin impregnation, then re-embed CELLOIDIN EMBEDDING Celloidin embedding is a slow process, usually taking weeks, and does not produce sections as thin as those produced by paraffin embedding. The advantage of celloidin embedding is that it completely avoids the use of heat at any stage. As a consequence, heat produced artifacts are avoided. In particular, shrinkage is absolutely minimal, if there is any, and structural relationships of the various types of tissue components can be seen clearly. The disadvantages are the longer time to cut, the thickness of the sections, the necessity for staining to be done on free floating section, the inconvenience of having to store the blocks in sealed jars with tight lids to prevent complete evaporation of 70% ethanol, the resulting restrictions on the type of staining methods that may be used. Celloidin may be purchased either as a solution or as a solid, damped with a liquid (usually ethanol) to reduce flammability. The stock purchased as a solution may be in an undesirable solvent and it is often the practice to evaporate the solvent to obtain dry celloidin, which is then weighed and re-dissolved in the appropriate solvent. Celloidin is used in form of solution, usually in a 1:1 mixture of ethanol-ether at concentrations of 2%, 4% and 8%. The fastest way to dissolve celloidin is to soak it first in half the final volume of anhydrous ethanol to soften it (50 mL for each 8 grams celloidin) with intermittent mixing in a tightly stoppered container. The next day, an equal volume of diethyl ether is added and intermittently mixed until an evenly consistent solution is obtained. The 2% and 4% solutions may then be made by simple dilution of the 8% solution with an equal parts mixture of ethanol and diethyl ether. The evaporation to dryness is done slowly at room temperature, without additional heat and in an explosion safe environment as a fire safety precaution. The solution should be transparent, without undissolved material, and should be stored in a completely closed container which is ether resistant. For delicate tissues, gradual dehydration with several changes of alcohol is strongly recommended to avoid distortion from removing the water too fast. No clearing agent is used with celloidin and following dehydration with absolute ethanol, the tissue may be placed in ethanol-ether. Ether is a lipid solvent and will remove