Prior to LCM, cut tissue samples are fixed, stained, and dehydrated if necessary. Authors use classical HE staining for visceral and lymph node tissues. Once the slice is ready, LCM should be performed within 45 min. All reagents, tubes, and other materials for RNA extraction should be ready before staining. This experiment was derived from PCR Laboratory Guide (2nd edition) by Seed Kang and Qu Lijia.
Operation method
HE staining of slices for LCM
Materials and Instruments
Ethanol Eosin Y Meyer hematoxylin Ultrapure water Xylene Move I. Materials For more product details, please visit Aladdin Scientific website.
Conical tubes Tweezers Broken glass dye pots
1. buffers, solutions and reagents
100% ethanol
70% ethanol
Eosin Y (Sigma)
Meyer's Hematoxylin (Sigma)
DEPC-treated ultrapure water (BiotecxLaboratories, stored at 4°C)
Xylene (Sigma)
2. Special equipment
Conical tube, 50ml
Tweezers
Broken glass dye vat (for xylene holding)
3. Cells and tissues
Pieces from Option 1
II. Methods
Staining should be organized as tightly as possible between the staining and LCM operations, especially the H20 and the final step of the ethyl fermentation must be fresh. Always remember that the amount of degraded RNA is proportional to the time the sample is in the aqueous phase after fixation (it is better not to exceed 5 mm in order to get high quality RNA). Use DEPC-treated water in all steps. Ethanol and xylene in the final step may absorb moisture; if humidity is high, it is recommended to pass the slice 3 times in 100% ethanol and wash it for 2 min the second time when it is placed in xylene. if humidity is low, omit pure ethanol for the third time and shorten the time in xylene to 30 s. Wipe off the water from the edges and the back of the slice before proceeding to each next step. Sometimes the slice rejects the solution; repeat if necessary. Hold the slice with tweezers.
1. prepare the staining solution (Fig. 11-3).
2. immerse in 70% ethanol for 30s.
3. Wash in pure water (do not use PBS instead of water, if PBS is chosen it will result in poor adhesion between the tissue and the LCM membrane), and move the section up and down in the staining cylinder for 5~30s.
4. Mayer's hematoxylin (Mayer's Hematoxylin) 30s
5. Pure water wash (do not use PBS instead of water, if PBS is used, it will lead to poor adhesion between the tissue and the LCM pancreas), move the section up and down in the vat, 5~30s.
6. 70% ethanol wash for 30s.
7.100% ethyl moss wash for 30s.
8.Eosin Y in 30s.
9.100% ethanol wash 30s,repeat 2~3 times.
10.Xylene wash 0.5~2min, 2 times. At this time for LCM sample preparation is complete. 