Preparation of tissue sections
ã€purpose】
Tissue specimens must first be sliced into tissue and then stained for clinical diagnosis and scientific research under the microscope .
ã€principle】
Hematoxylin staining combined with eosin staining is a conventional histological staining technique, also known as H & E staining. Hematoxylin is alkaline staining, the nucleus is dyed dark purple or blue, and is often used for nuclear staining; eosin is acid stained, and the cytoplasm is stained red, which is often used for cytoplasmic staining.
ã€material】
1. Reagents and solutions
( 1 ) 2- methylbutanol ( isoamyl alcohol )
( 2 ) dry ice
( 3 ) OCT (Tissue-Tek, SAKURA)
( 4 ) Frozen or paraffin tissue blocks
( 5 ) Acidic Harris hematoxylin dye
( 6 ) 1 % acidic alcohol
70 % alcohol          99 ml
Concentrated hydrochloric acid             1 ml
( 7 ) alcohol soluble eosin dye
( 8 ) Alcohol
( 9 ) xylene
( 10 ) Resin sealing liquid
2. Equipment
( 1 ) plastic mold
( 2 ) Long scorpion
( 3 ) Superfrost Plus Slides (Fisher Scientific)
( 4 ) coverslips
( 5 ) blade ( one-time or non- One time ) ;
( 6 ) Frozen or paraffin slicer (Leica, Micorm or others )
ã€method】
Method 1 , preparation of frozen sections
First, prepare frozen tissue
1. Put 2- methylbutanol ( isoamyl alcohol ) in a plastic or stainless steel container , then put a small piece of dry ice from time to time to lower the temperature to -40 o C to -50 o C and keep it cool.
2. Mark the plastic mold and pour the OCT into the mold to cover the bottom.
3. Collect tissue specimens. Fast, gentle and accurate material to ensure the quality of tissue specimens is the most basic condition for obtaining high quality immunolabel staining.
4. Remove tissue specimen is placed into OCT filled with a mold, fill the mold with OCT, until the specimen is completely covered thereof. Tissue specimens should be placed as close as possible to the bottom of the mold so that the specimen is easily exposed during slicing. Adjust the location of the tissue specimen as appropriate. The mold was placed in a long tweezers and placed in a cooled solution of 2- methylbutanol ( isoamyl alcohol ) . To avoid cavitation, the first solution will touch the bottom mold, the mold filled with samples from the bottom to the surface of the frozen, and then the whole mold into the solution for 5-10 minutes.
( 1 ) Most of the removed tissue specimens can be placed directly into the mold. If the specimen is contaminated with a large amount of liquid, the excess liquid should be removed with a paper with good water absorption properties and then frozen and embedded. Do not wash the specimen with a solution.
( 2 ) Tissue specimens that need to be fixed first can be treated with 10 % to 30 % sucrose - buffer after freezing, and then frozen and embedded.
5. Store frozen tissue specimens in a -80oC refrigerator for use.
Second , making frozen slices
1. Remove the frozen tissue specimen from the -80oC refrigerator before slicing and place it in a frozen slicer (-20oC) for rewarming.
2. Use OCT to freeze the frozen tissue block onto the frozen specimen plate and secure it to the microtome. After adjusting the position of the tissue block plane and the blade, start the slice. After the cut is made, the slice is collected. Slices can be selected to different thicknesses depending on the purpose of the study. 3-6 μm is a commonly used slice thickness or 25-50 μm thick.
3. Sections are allowed to dry at room temperature and then fixed with the desired fixative. If cold acetone or acetone / methanol is used , the sections are fixed after 20 minutes of drying and allowed to dry at room temperature, or they can be used for dyeing directly or placed in a sealed slice box and stored in a -80oC refrigerator for use.
4. The rest of the frozen tissue block, OCT can be used to cover its exposed face and then stored in a refrigerator at -80ºC until the next use.
Method 2 , preparation of paraffin sections
1 . Collect tissue samples and fix them. 10 % formalin buffer is the most commonly used fixative, and other fixatives can be used according to the needs of the experiment. According to the size of the specimen, it is fixed at room temperature for 8-24 hours, or longer. The thickness of the specimen is preferably not more than 3 mm . After the fixation is completed, the specimen can be processed in the next step.
2 . The production of paraffin tissue blocks requires specialized equipment as well as complex tissue processing. Usually done by a histology or pathology laboratory.
3 . Paraffin section. Prepare a water bath containing 40oC distilled water, fix the wrapped paraffin block on the paraffin slicer, cut into a certain thickness ( usually 4-6 μm) as needed , float the slice on the water surface of the water bath, and then turn Go to the Superfrost Plus film. The sections were naturally air dried overnight at room temperature and stored for later use.
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