Preparation Method for Paraffin-embedded Tissue Sample
After tissue collection, please perform fixation, storage, paraffin embedding, section, water bath, bake and then HE staining (or other chemical dyes) and immune antibody labeling.
1. Tissue Collection, Fixation and Storage
According to experimental purposes and tissue characteristics, different fixation and storage methods can be adopted. Here, we only introduce the commonly used methods.
After the tissue collection, use 4% paraformaldehyde (4%PFA) to fix for 1 hour at 4°C (adjust fixation time based on the tissue size and density) or overnight, wash three times by using PBS buffer solution from 10 minutes to 1 hour for each time. Place into 30%, 50% ethanol respectively for 10 minutes to 1 hour and store in 70% ethanol at 4°C.
The fixed sample undergoes gradient ethanol dehydration, xylene lucency, paraffin embedding at 52-54°C, and regular section with 4-10mm. Paste it on the processed and clean glass slide, bake the slide at 34°C overnight and collect into slide box with sealing preservation at 4°C.
Paraffin-embedding
Tissue sample is stored in 70% ethanol at 4°C
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80% Ethanol 15min
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95% Ethanol 15min
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100% Ethanol 15min X 2
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1/2 Ethanol 1/2 Xylene 15min
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Xylene Lucency 5-10min
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1/2 Xylene 1/2 Paraffin 30min
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Paraffin (1) 1.5hr
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Paraffin (2) 1.5-2.5hr
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Paraffin (3) Embedding
After the section undergoes xylene dewaxing and gradient ethanol rehydration, perform immune response. Instructions are the same as the frozen tissue section.
Dewaxing and rehydration for paraffin tissue section:
Tissue section sample is sealed storage at 4°C
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Xylene (1) 10min
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Xylene (2) 10min
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Xylene (3) 10min
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1/2 Ethanol 1/2 Xylene 15min
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100%Ethanol 5min
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1/2 Xylene 1/2 Paraffin 30min
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95% Ethanol 5 min
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80% Ethanol 5 min
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70% Ethanol 5 min
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50% Ethanol 5 min
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30% Ethanol 5 min
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d2H2O 5min
After the section undergoes xylene dewaxing and gradient ethanol rehydration, perform immune response. Instructions are the same as the frozen tissue section.
Dewaxing and rehydration for paraffin tissue section:
Tissue section is sealed storage at 4°C
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Xylene (1) 10min
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Xylene (2) 10min
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Xylene (3) 10min
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1/2 Ethanol 1/2 Xylene 15min
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100% Ethanol 5min
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95% Ethanol 5min
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80% Ethanol 5min
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70% Ethanol 5min
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50% Ethanol 5min
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30% Ethanol 5min
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d2H2O 5min
After tissue section undergoes xylene dewaxing and gradient ethanol rehydration, stain it in hematoxylin for 5-20 minutes at room temperature and quickly wash the section till the tissue section is becoming blue with microscopy. Use 0.1-0.5% hydrochloric acid in ethanol for color separation for 1~2 seconds, or use 50ml H2O with one drop of ammonia water to obtain the same effects (if the staining is perfect, you may skip color separation). Wash the tissue section by using running water till the section is becoming blue. Tissue section undergoes the gradient ethanol to 95%, stain in 0.5% eosin for 1~5seconds or even longer time and wash the residual color by using 95% ethanol. If the color is too dark, it can be decolorized in ethanol with low concentration with microscopy. Use absolute alcohol for dehydration, xylene for lucency and seal section with neutral balsam. Observe results by using ordinary light microscope and store stained section in a dry at room temperature.
Xylene Dewaxing and Gradient Ethanol Rehydration for Paraffin Section
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Hematoxylin 5-10min at Room Temperature
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H2O Quick Wash
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0.1% Hydrochloric Acid in Ethanol 1-2sec
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Drip Wash by using Running Water
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30% Ethanol 1min
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50% Ethanol 1min
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70% Ethanol 1min
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80% Ethanol 1min
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95% Ethanol 1min
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Eosin A Solution 1min
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Eosin B Solution 1-30sec
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Absolute Alcohol 1min
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Absolute Alcohol 5min X 2
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1/2 Ethanol 1/2 Xylene 15min
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Xylene (1) 5min
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Xylene (2) 5min
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Xylene (3) 5min
Seal section with neutral balsam and store in a dry place at room temperature
