|
|
Page designed
and maintained by Octavian Henegariu (Email:
or ). |
|
WARNING:
The information provided in these pages is copyrighted and is intended
for individual use only. No parts of this work (text, tables or
pictures) may be commercialized, published or otherwise reproduced
without the written consent of the author. |
FISH on paraffin embedded tissue
(PET)
TOPICS:
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| 12
| 13
| 14
| 15
| 16
| 17
| 18
| 19
| 20
| 21
| 22
| 23
FIGURES: | 1
| 2
|
3 |
4 |
5 |
6 |
7
| 8
| 9
| 10
| 11
| TABLES:
| 1
| 2
| 3
| 4
| 5
| 6
| 7
1. Using DNA probes
-
In our experience, probably the most important step for FISH on PET was slide pretreatment with proteases. Paraffin embedded material required much higher concentrations of pepsin (0.4-5%) compared to 0.005% for regular cytogenetic preparations. Protease digestion can be done between 2 and 20 minutes, depending on how the tissue was fixed. Hybridizaton results are influenced by the original fixation procedure, and some paraffin sections may not hybridize well.
- Protocol: alternatively, and probably with even better success, one can use Proteinase K to treat the deparaffinized sections prior to hybridizatoin. Briefly, the slide with the paraffin section is placed in a jar with xylene for 10-15 minutes, then in a jar with 1:1 xylene:ethanol for 10 minutes, followed by incubation only in 100% ethanol for 10 minutes. A ProteinaseK digestion solution is prepared by mixing 20ml PBS + 100µl 10%SDS + 200µl 20mg/ml Proteinase K. We do the digestions in a small plastic slide holder, which accomodates 20 ml liquid. Other jars can be used as well. The jar with the proteinse solution is kept in a waterbath at 45-50 C. The deparaffinated slides are incubated in the proteinase K solution 8-25 minutes,depending on how the paraffinization and fixation was done. The optimal incubation time needs to be identified by parallel experiments. After digestion, the slide is rinsed 1-3 minutes in PBS, followd by 5 minutes incubations in 70% and 100% ethanol. No further fixation is needed. Slides can be denatured and used. This protocol worked equally well for DNA and RNA probes !!.
-
Hybridizations on PET also benefit from longer denaturing times (5-6 minutes) and
-
higher denaturing temperatures (up to 82-85 C).Denaturing is done in 70%FA/2xSSC.
-
Another parameter is the thickness of the section. Whereas for RNA hybridizations 2-3 µm sections were ideal, DNA hybridizations worked best on 10µm thick sections. This correlates with the RNA being found primarily in the cytoplasm, where it is readily available to hybridizations, whereas the DNA is in the nucleus. Sections that are too thin may include only thin areas/slices of the nuclei, which may lack the targeted sequences and thus will provide false negative results.
In FISH on PET it is very important that the DNA probe used is cut in small fragments (200bp average). Too long DNA fragments will not penetrate the tissue and will not reach the target DNA. Posthybridization washes should be gentle, as the probe fragments are small and the tissue is "hard" to hybridize onto. Too stringent washes may wash away specific signals.
An example of triple color FISH with DNA painting probes on PET is provided in Fig.11l (courtesy of Dr. Ronald Honchel) where three 12p specific paint probes were used on a PET section of a germ cell tumor tissue. Many nuclei show more than two groups of signals, indicating an increase in 12p copy-number , a common feature of germ cell tumors.
2. Using RNA probes (to target gene expression)
Slide preparation and preatreatment protocol is the same as above. We used riboprobes and double-stranded DNA probes (gene exons generated and labeled by PCR) with equal success. However, it is important to mention that the majority of RNA signals were too weak to be visualized using fluor-dUTP or fluor-UTP labeled probes. Instead, we labeled the riboprobes or DNA probes with haptenes (BIO-, DIG-, DNP-dUTPs or -UTPs) and detected them with fluor-labeled antibodies. This approach was always more efficient. Also successful was the use of antibodies against fluorophores (antibodies against fluorescein and rhodamine derivatives can be used to amplify the signals as well).
An example of RNA in situ detection is shown in the figure below. (1) RNA detection. DIG-labeled riboprobes of the rat surfactant protein B (SPB) were detected red (upper left image) in the type II pneumocytes in the lung alveoli. On a different slide (upper right image), SPB expression was also detected (green) in the cells lining the lumen of small bronchioli. (2) RNA and DNA detection. Using a mouse chromosome Y-specific probe (labeled red) and two small DNA probes (labeled green) for three exons of the mouse gene for albumin, we showed that a female mouse, recipient of a bone marrow transplant from a mouse male donor, carried male cells fully differentiated into hepatocytes. In all images, the DNA was counterstained with DAPI (blue). The red arrow shows the Y-specific centromere signal (red) in one of the nuclei, whereas the green arrows show the abundent hybridization of the two exon-type DNA sequences (each ~100 bp long ) in the cytoplasm of most hepatocytes, and also in their nuclei (the nuclear signals are not from the genomic DNA sequence of the albumin gene, but rather from the mRNA transcription centers). In multinuclear hepatocytes, 4-8 such strong nuclear green signals were clearly visible. The short blue arrow points to a nucleus which contains both the Y-signal and the albumin transcription centers, and is surrounded by abundedent mRNA signal in the cytoplasm.
