Preparation Method for Frozen Tissue Section Sample

After tissue collection, please perform fixation, cryopreservation, section, dry and then immune antibody labeling.

1. Tissue Collection, Fixation and Storage

According to experimental purposes and tissue characteristics, different fixation and storage methods can be adopted for tissues from various sources. Here, we only introduce the commonly used methods.
1) Liquid Nitrogen Freezing Method
Collect the fresh tissue and put into liquid nitrogen for preservation.
2) Fixation
After the fresh 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 with 10-30 minutes for each time. Immerse into 20% sucrose for 1 hour or overnight and store in 20% sucrose (containing 0.05%NaN3) at 4°C.

The freezing methods for tissues are based on the various fixation and storage conditions. Use freezing microtome to cut frozen tissue into 4-20μm sections, paste on the processed glass slide and make them dry for 1 hour at room temperature. Now, you can perform immunolabeling or place into slide box with sealing preservation at -80°C.
1) Gas CO₂ Freezing Method:
Place the sample in the OCT of the sample stage, open the cylinder switch to release CO₂ and quickly freeze tissue sample.
2) Dry Ice Therapy: (suitable for tissues with small blocks and low density, such as embryo, small piece of tissue, biopsy, etc)
Put the small tissue blocks into frozen-embedding mold, adjust the section position and place them on the dry ice (if no dry ice, then store at -80°C). Keep them sealed at -80°C after the complete freezing.
3) Direct Freezing Method
Directly paste the sample on the sample stage with OCT and cryopreserve in freezing microtome (box temperature: -80°C).

Take the frozen tissue section out of -80°C refrigerator and let it stand for 10 minutes at room temperature. When the section restores to normal temperature and becomes dry, elute OCT by using PBS for 10 minutes, and perform chemiluminescence labeling without Triton treatment. Please refer to the kit for instructions.

4. Immunofluorescent Labeling for Frozen Tissue Section

Take the frozen tissue section out of -80°C refrigerator and let it stand for 10 minutes at room temperature. When the section recovers to normal temperature and becomes dry, elute OCT by using PBS for 10 minutes, and perform immune antibody labeling without Triton treatment. Please refer to immunolabeling method for instructions.

1) Alkaline Phosphatase (AP):

Tissue Section with Sealed Storage
PBS  5min
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Seal in PBS containing 0.5%NS,1%BSA for 30min-1hr at Room Temperature
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Primary Antibodies (diluted) 1-1.5hr at Room Temperature or 4°C Overnight
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PBS  10min  X 3
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Secondary Antibodies-AP (diluted) 1.5-2hr at Room Temperature
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PBS  10min  X 3
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Coloration: AP-CDS  5min X 2
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NBT4.5ml/ml, BCIP3.5ml/ml in AP-CDS  at Room Temperature or 37°C 5-30min
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TE 10min at Room Temperature
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Gradient Ethanol Dehydration

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Xylene Lucency
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Seal Section with Neutral Balsam

Tissue Section with Seal Preservation
PBS  5min
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3%H₂O₂.10% MeOH in PBS  3min at room temperature
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PBS  5min X 3
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Seal in PBS containing 0.5%NS,1%BSA for 30min-1hr at room temperature
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Primary Antibodies (diluted) 1-1.5hr at Room Temperature or 4°C Overnight
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PBS  10min  X 3
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Secondary Antibodies-HRP (diluted) 1.5-2hr at Room Temperature
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PBS  10min X 3
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Coloration: HRP-CDS 20-30min at Room Temperature
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Gradient Ethanol Dehydration
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Xylene Lucency
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Seal Section with Neutral Balsam