Five Types of Tissue Preparation for Immunohistochemistry

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Abstract:    Tissue preparation of immunohistochemical reaction has been mentioned in the previous article about immunohistochemistry, which greatly determines whether the experiment can be successful. Let's learn details and methods of five kinds of tissue preparation.
Keywords: Tissue Preparation for Immunohistochemistry, IHC

1. Slides in Tissue Preparation

High quality immunohistochemical result requires the reasonable combination of tissue slices and slides in order to prevent reagent aggregations and pseudostaining caused by tissue adhesion. Commercial slides specially used to perform immunohistochemical staining are standardizedly coated. Please find two coating protocols for regular slides used in immunohistochemical staining.

1.1. Poly-L-Lysine Coating Slides Protocol

  • Prepare 0.1%(w/V) poly-l-lysine solution(Sigma P-8920);
  • Immerse the slide in the solution;
  • Dry at room temperature;
  • Store at room temperature.

1.2. APTS Coating Slides Protocol

  • Prepare acetone solution containing 2%(V/V) silane(Sigma A-3648);
  • Immerse the slide in the acetone for 1-5min;
  • Immerse the slide in the acetone solution containing 2% silane for 1-5min;
  • Continuously wash the slide in the acetone solution twice for 5min;
  • Dry and store at room temperature.

2. Preparation of Cell Smear/Centrifuged Cell Smear

  • Prepare cell scraping smear/centrifuged cell smear with slide/poly-l-lysine coating slides;
  • Dry at room temperature for 30min;
  • Fix in pre-cooling and fresh acetone for 20min at -20℃;
  • Air-dry at room temperature for 15min at least;
  • Stain cell scraping smear/centrifuged cell smear.

3. Frozen Sections in Tissue Preparation

  • Cut four 7μm unfixed frozen sections;
  • Put sections on slide/poly-l-lysine coating slides;
  • Air-dry sections in the air for 30min;
  • Put sections in fresh acetone pre-cooled to 4℃ and fix for 20min;
  • Air-dry sections for at least 15min at room temperature;
  • Stain sections.

4. Notes for Cell Smear and Frozen Sections

  • Fresh acetone can be replaced by 100% ethanol; The selection of fixative depends on the target antigen to be stained in the tissue;
  • Unstained slides can be coated in aluminum foil and stored at -20℃. When ready for staining: open and recover aluminum foil to room temperature. Fix for at least 30s with the same fixative before the storage. Air-dry slices and then rehydrate for 5min in buffer. Finally the staining is performed;
  • The stability of unstained slides depends on characterization of antigens; Some antigens are very unstable and should be stained after smearing.

5. Paraffin Sections

  • Fix the tissue in 10% formaldehyde for 12-48h(other fixatives may be also suitable.) Fixation time depends on the thickness and temperature of tissues as well as properties of antigens ready for detection;
  • Paraffin-embedded;
  • Cut 3-5μm section;
  • Put the section on slide/poly-l-lysine coating slides and perform the water bath without the adhesive;
  • Blot up the water completely along the edge of the section;
  • Put the section in 60℃ oven for 30-60min;
  • Stain the section after being taken off paraffin.

Notes

  • Extending the drying time(e.g. Overnight at room temperature)for tissues rich in fat(e.g. brain, breast) may enhance the adhesion of tissue slices;
  • Over 60℃ drying temperature may be harmful to the detection of some antigens or increase background staining;
  • In any storage condition, extending the storage time of paraffin section may decrease immunoreactivity of some antigens. The following antigens are easy to be degraded: estrogen receptor, progesterone receptor, proteins related to cell cycle regulation(e.g. Ki-67、PCNA、cyclinD1、bcl-6) and thyroid transcription factor-1. It's unsuitable to store paraffin sections for a long time;
  • If the staining of the archival paraffin section is inconsistent, fresh sections should be used to repeat the experiment.

6. Cell Block

This step is suitable for the difficult paraffin embedding because the tissue block is too small. A reagent that forms so-called cell blocks is essential. Four different types of preparing cell blocks are below.

6.1. Agar Cell Block Preparation

  • Take 20ml precipitation sample at the bottom and perform the centrifugation at 3000r/min for 5-10min;
  • Blot up the supernatant and fix centrifugal sediments for 1h by 10% neutral formalin;
  • Blot up the liquid;
  • Add a little melted agar and shake the test tube for the complete mixture. Then perform the centrifugation at 3000r/min for 5-10min. At present, agar has been solidified;
  • Take out the agar cell block and put it into dehydration box with the suitable cutting size. Then prepare paraffin-embedded blocks as usual.

6.2. Albumen Cell Block Preparation

  • Take 20ml precipitation sample at the bottom and perform the centrifugation at 3000r/min for 5-10min;
  • Add lml egg white and then gently scrape cells at the bottom of the tube with flat bamboo stick, making cells form the cluster and suspended in egg white. Similarly, perform the centrifugation at 2000r/min for 5min;
  • Remove redundant egg white and gently add 2ml 10 % neutral formalin fixative solution along the tube wall. The egg white is immediately solidified to get cell cluster. Then, shake gently to make cell cluster suspended and fixed in the fixative solution overnight. Gently take out the cell cluster and wrap it with the embedding paper. Finally, put it into dehydration box and prepare paraffin-embedded blocks as usual.

6.3. Direct Cell Block Preparation(Applicable for A Large Amount of Cell)

  • Take 20ml precipitation sample at the bottom and perform the centrifugation at 3000r/min for 5-10min;
  • Blot up the supernatant. Then, gently add 95% alcohol along the tube wall and perform the centrifugation at 3000r/min for 5min. The standstill time should be no less than 1h;
  • Gently take out sediments with a bamboo stick and wrap it with the embedding paper;
  • Put it into dehydration box and fix it by 10% neutral formalin for 1h. Then prepare the paraffin section as usual.

6.4. Glutaraldehyde Cell Block Preparation

  • Take 20ml precipitation sample at the bottom and perform the centrifugation at 3000r/min for 5-10min;
  • Blot up the supernatant. Add 10ml 3% glutaraldehyde for the complete mixture. Then, perform the centrifugation at 3000r/min for 5-10min;
  • Gently take out sediments with a bamboo stick;
  • Put it into dehydration box and fix it by 10% neutral formalin for 1h. Then prepare the paraffin section as usual.