Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Personalized medicine requires the different types of analysis from classical histology to molecular analysis, and various in-situ techniques are performed on a tumor to select the best therapy. The investigative techniques allow various analytical technologies from a single specimen. We demonstrate here, by using a non-cross-linking fixative in the simple formalin post-fixation step that you may use protocols originally developed and validated for formal and fixed paraffin embedded material, can be used from the cell sample yielding good morphology, good RNA quality, and good results for fuller sense in-situ hybridization.

The typical hybridization conditions are not appropriate for formalin-free fixed tissues to generate FISH results. We revealed that it is not the penetration but the chemical correction time that is critical to achieve comparable FISH results from pectin-fixed tissue. Only the section used for FISH is formal and post fixed.

The remaining formalin-free fixed tissue enables superior molecular analysis relative to FFP tissue, as demonstrated by RTPCR. The procedure that we demonstrated by by Daniela Pabst, the technician from my laboratory. To begin, cut the breast cancer tissue samples with a sterile scalpel into two halves, each four millimeters thick to ensure adequate tissue quantity.

Use a maximum size of 4x15x15 millimiters for fixation through standard buffered formalin solution, or through alternative non-cross-linking, formalin-free tissue fixation. Place one half of the tumor specimen in a standard tissue cassette, and submerge it in SBFS. Place the other half of the tumor specimen in the alternative fixation solution for 24 hours with a volume-per-volume ratio of four parts fixative and one part tissue.

Then, transfer the alternatively fixed samples into the stabilizer solution for two to 72 hours by opening the container, turning the tissue cassette by 180 degrees, and submerging it. Next, place the tissue cassettes with the SBFS-fixed samples in a container filled with 70%ethanol for 30 minutes at room temperature to remove residual SBFS. Following tissue processing and embedding as described in the text protocol, use a microtome to cut three micron sections of FFPE and NCFPE blocks.

Place some of each type of section in a cold water bath. Pick up the floating section with a fine paintbrush on a fine microscope slide, and transfer it into a warm water bath. Pick up the stretched sections again with the paintbrush onto the microscope slide without any wrinkles.

Also prepare for RNA isolation to perform reverse transcription PCR on the same sample by placing some of each type of section into a reaction vial. For deparaffinization of tissue sections, we hydrate the sections stepwise at room temperature in the staining jars with 250 milliliters of different reagents. First place the sections in 100%xylene two times for 15 minutes, then transfer the sections to 96%ethanol two times for 15 minutes.

Next, move the sections through incrementally lower percentages of ethanol, incubating each solution for two minutes. Finally, transfer the sections to distilled two times for 10 minutes. Perform post-fixation of NCFPE slides by putting them into a new staining jar filled with SBFS for 24 hours at room temperature.

Leave the jar in the fume hood during the 24 hour post-fixation step. Surprisingly, all the post-fixation times of longer than 18 hours gave results similar to that obtained with formal and fixed paraffin material. Remove the excess SPFS from the slides by washing them in phosphate buffered saline for 10 minutes three times.

Follow with the second wash in distilled water for 10 minutes two times. Next, place the staining jar containing 250 milliliters of heat pre-treatment solution citric in a 98 degree celsius water bath. Place the hydrated FFPE in post-fixed NCFPE slides into the jar, and incubate for 15 minutes.

Then, wash the slides in distilled water for three minutes at room temperature in a new staining jar. For proteolysis, place the slides into a temperature controlled hybridization at 37 degrees celsius. Immediately apply the ready to use pepsin solution dropwise to the tissue sections until they are completely covered.

Then, incubate for nine minutes at 37 degrees celsius in the hybridization system. Next, place the slides into a jar containing SSC wash buffer, and incubate for five minutes. Place the slides in distilled water for one minute at room temperature.

Perform dehydration of the sections in a series of graded alcohols, each for one minute. Subsequent to the dehydration series, air dry the sections. For hybridization, pipette 10 microliters of the combined HER2 and sun 17 probe onto the dried tissue sections, cover each sample with a cover slip, and seal it with rubber cement.

Code denature the probe and specimen DNA in the hybridization system at 75 degrees celsius for 10 minutes. The hybridization system automatically decreases to 37 degrees celsius, and hybridization goes on overnight. The next day, carefully remove the rubber cement and the cover slips as well with forceps for post-hybridization and protection.

Incubate the sizes in a staining jar containing 37 degrees celsius pre-warmed 1X buffer A for five minutes. Next, wash the slides using pre-warmed 1X wash buffer twice for five minutes at 27 degrees celsius. Then dehydrate the slides in 70%90%and 100%ethanol at one minute for each concentration.

Dry the samples in air, and protect them from light. For staining, apply 30 microliters of DAPI-solution to the hybridized area, avoiding bubbles. Cover the sections with a cover slip before incubating the sections for 15 minutes, protected from light.

Carefully remove excess DAPI-solution by gently pressing the slide between filter papers. Store the slides at two to eight degrees celsius in the dark for up to two weeks until evaluation by confocal microscopy. Acquire the images with filters for green and orange channels.

Proceed to data interpretation and assessment of RNA quality as described in the text protocol. Shown here is the direct comparison of the result obtained from the formal and fixed sample, and the non-cross-linking fixed sample that has been post-fixed. The signals are of similar intensity, and the background is also similar.

In normal interphase cells, there are an equal number of red and green signals corresponding to the HER2 locus and the sen 17 reference locus which shows that there is no amplification at the HER2 gene. Shown here is the result of another case where the HER2 gene is amplified, and there is a much stronger and wider signal for the HER2 locus than for the reference gene. This phenomenon is apparent in the formal and fixed, and the non-cross-linking fixed tissue.

This graph shows the amplification efficacy for the different amplicon lengths of the GAPDH mRNA. For formalin, about 23 cycles are necessary, and for non-cross-linking fixed material, this is decreased to 20 cycles. The difference becomes more evident at longer amplicons which indicates that both fragmentation and chemical modification are markedly reduced by using non-cross-linking fixatives.

After watching this video, you should have a good understanding how one can use an FDA approved key to for FFPN material on non-cross-linking fixed material by adding a simple post-fixation step in the pre-analytic procedure. The main advantage of this technique is that only the section used for full sense in-situ hybridization is for post-fixed with formalin, whereas the remaining block is fixed in the non-cross-linked fixative, and therefore can be used for a broad spectrum of molecular analysis. This protocol is particularly useful in the context of modern personalized medicine when multiple analyses have to be performed from relatively small tumors, and processing tissue specimens for different protocols is not feasible.

Furthermore, using a non-cross-linking fixative has the advantage that it preserves tissue properties like cryopreservation. This avoids the need for freezing liquid nitrogen, and to maintain a cooling chain in a chemical context. These differences in RNA quality are not obvious, so that when using classical RNA quality control assays such as RNA integrity numbers, since these assays are not fully formative in RNA quality isolated from fixed and paraffin-embedded material.