Hydatidiform moles are abnormal human pregnancies with heterogeneous aetiologies that can be classified according to their morphological features and parental contribution to the molar genomes. Here, protocols of multiplex microsatellite DNA genotyping and flow cytometry of formalin-fixed paraffin-embedded molar tissues are described in detail, together with results’ interpretation and integration.
Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are two types of HM based on microscopic morphological evaluation, complete HM (CHM) and partial HM (PHM). These can be further subdivided based on the parental contribution to the molar genomes. Such characterization of HM, by morphology and genotype analyses, is crucial for patient management and for the fundamental understanding of this intriguing pathology. It is well documented that morphological analysis of HM is subject to wide interobserver variability and is not sufficient on its own to accurately classify HM into CHM and PHM and distinguish them from hydropic non-molar abortions. Genotyping analysis is mostly performed on DNA and tissues from formalin-fixed paraffin-embedded (FFPE) products of conception, which have less than optimal quality and may consequently lead to wrong conclusions. In this article, detailed protocols for multiplex genotyping and flow cytometry analyses of FFPE molar tissues are provided, along with the interpretation of the results of these methods, their troubleshooting, and integration with the morphological evaluation, p57KIP2 immunohistochemistry, and fluorescence in situ hybridization (FISH) to reach a correct and robust diagnosis. Here, the authors share the methods and lessons learned in the past 10 years from the analysis of approximately 400 products of conception.
A hydatidiform mole (HM) is an abnormal human pregnancy characterized by abnormal embryonic development, hyperproliferation of the trophoblast, and hydropic degeneration of chorionic villi (CV). Historically, HM used to be divided into two types, complete HM (CHM) and partial HM (PHM) based only on morphological evaluation1. However, it has been shown that morphological evaluation alone is not sufficient to classify HM into the two subtypes (CHM and PHM) and distinguish them from non-molar miscarriages2,3,4.
Because CHM and PHM have different propensities to malignancies, it is therefore important to accurately determine the genotypic type of HM to provide appropriate follow-up and management to the patients. Consequently, in the past decades, several methodologies have been developed and evolved for the purpose of identifying the parental contribution to the molar tissues and reaching a correct classification of HM. These include karyotype analysis, chromosomal banding polymorphism, human leukocyte antigen (HLA) serological typing, restriction fragment length polymorphism, variable number of tandem repeats, microsatellite genotyping, flow cytometry, and p57KIP2 immunohistochemistry. This has allowed accurate subdivision of HM conceptions based on the parental contribution to their genomes, as follows: CHM, which are diploid androgenetic monospermic or diploid androgenetic dispermic, and PHM, which are triploid, dispermic in 99% and monospermic in 1% of the cases5,6,7,8. Furthermore, there is another genotypic type of HM that emerged in the past two decades, which is diploid biparental. The latter is mostly recurrent and may affect a single family member (simplex cases) or at least two family members (familial cases). These diploid biparental moles are mostly caused by recessive mutations in NLRP7 or KHDC3L in the patients9,10,11,12. Diploid biparental HM in patients with recessive mutations in NLRP7 may be diagnosed as CHM or PHM by morphological analysis and this appears to be associated with the severity of the mutations in the patients13,14. In addition to the classification of HM according to their genotypes, the introduction and use of several genotyping methods allowed the distinction of the various molar entities from non-molar miscarriages, such as aneuploid diploid biparental conceptions and other types of conceptions5,15. Such conceptions may have some trophoblast proliferation and abnormal villous morphology that mimic, to some extent, some morphological features of HM.
The purpose of this article is to provide detailed protocols for multiplex genotyping and flow cytometry of formalin-fixed paraffin-embedded (FFPE) tissues, and comprehensive analyses of the results of these methods and their integration with other methods for correct and conclusive diagnosis of molar tissues.
This research study was approved by the McGill Institutional Review Board. All patients provided written consent to participate in the study and to have their FFPE products of conception (POCs) retrieved from various pathology departments.
NOTE: While there are several methods for genotyping and ploidy determination by flow cytometry, the protocols provided here describe one method of analysis using one platform for each.
1. Genotyping
Reagent | Quantity |
Eosin Y stock solution (1%) | 250 mL |
80% Ethanol | 750 mL |
Glacial Acetic Acid (Concentrated) | 5 mL |
Table 1: Eosin Y working solution (0.25%) preparation.
Reagent used (100 mL per bin) | Duration |
1) Xylene | 5 min |
2) Xylene | 5 min |
3) 100% Ethanol | 2 min |
4) 95% Ethanol | 2 min |
5) 70% Ethanol | 2 min |
6) 50% Ethanol | 2 min |
7) Distilled water | 5 min |
8) Hematoxylin | 4 min |
9) Distilled water | 5 min |
10) Eosin | 1 min |
11) 95% Ethanol | 5 min |
12) 100% Ethanol | 5 min |
13) Xylene | 5 min |
14) Xylene | 5 min |
Table 2: Reagents and durations for the H&E staining protocol.
Figure 1: Representative slide for genotyping. Top: A slide that needs to be "cleaned" to become free of maternal tissues. Bottom: The same slide shown after it has been cleaned and now contains nothing but CV for DNA extraction. Please click here to view a larger version of this figure.
Figure 2: Representative slide for genotyping. Top: A slide that needs to be "cleaned" to become free of maternal tissues. Bottom: The same slide shown after it has been cleaned and now contains nothing but CV for DNA extraction. Please click here to view a larger version of this figure.
Figure 3: Representative gel for DNA quantification. Included are the concentrations of each DNA, as measured using a spectrophotometer, and the quantities used for the multiplex PCR. Please click here to view a larger version of this figure.
Figure 4: PCR cycle conditions for the multiplex STR system. Please click here to view a larger version of this figure.
Figure 5: Screenshot showing the Size Standard Editor. Please click here to view a larger version of this figure.
2. Flow Cytometry
Figure 6: H&E section representing a POC block that is ideal for flow cytometry. Please click here to view a larger version of this figure.
Figure 7: H&E section representing a more difficult block for flow cytometry. This representative H&E section shows that only the bottom half of this section should be used for flow cytometry analysis, with the goal of enriching for the CV. The outlined area, labelled "CV," is mostly made up of CV. Please click here to view a larger version of this figure.
Reagent used (6 mL each) | Duration |
1) Xylene | 2 x 10 min |
2) 100% Ethanol | 2 x 10 min |
3) 95% Ethanol | 10 min |
4) 70% Ethanol | 10 min |
5) 50% Ethanol | 10 min |
6) Distilled water | 2 x 10 min |
Table 3: Reagents and durations for deparaffinization and rehydration.
Figure 8: Screenshot displaying a histogram and a dot plot of a representative sample that is ungated (A) and gated (B). Please click here to view a larger version of this figure.
The complexity of molar tissues and the fact that they may have various genotypes necessitates stringent analysis and the use of several methods such as morphological evaluation, p57 immunohistochemistry, microsatellite genotyping, flow cytometry, and FISH. For example, one patient (1790) was referred with two PHM that were found to be triploid by microarray analysis of the POCs only. The patient was therefore diagnosed with recurrent PHM. Microsatellite genotyping of her two "PHM" along with the DNA of the patient and her partner revealed that while the patient's first mole is triploid dispermic (Figure 9A), her second "PHM" has a triploid digynic genotype (Figure 9B) and is therefore not a PHM, but a non-molar miscarriage.
The first marker in Figure 9A (in black) shows two peaks in the POC. The first peak originates from the mother, since only the mother has a peak of this size. Following the same reasoning, the second peak originates from the father since he shares the same allele. Notice how the second peak is a lot higher than the first, indicating that there are probably two doses of the same paternal allele in that peak. Maternal contamination, which will be explained in more detail later on, is very minimal in this POC because the POC displays a very tiny peak at the position of the second maternal allele.
The second marker in Figure 9A (in blue) shows three peaks in the POC. Two of these peaks originate from the father and one from the mother. Thus, it is apparent again from this marker that there are three alleles present in the POC, two from the father and one from the mother. The third and fourth markers in Figure 9A are similar to the first marker, and also show two alleles coming from the father and one from the mother.
Since all four markers consistently show three alleles (by dose or by the presence of three alleles of different sizes), two originating from the father and one from the mother, one can conclude that this POC is triploid dispermic, and confirms the diagnosis of PHM.
All four markers in Figure 9B again show three alleles: the first marker shows two doses in the first peak that originate from the mother and one dose in the second peak that originates from the father. The second and fourth markers show three different peaks (i.e., three different alleles), two of which are from the mother. The third marker shows one dose in the first peak originating from the father and two doses in the second peak originating from the mother. Therefore, this POC is triploid digynic in origin since two sets of chromosomes come from the mother and one set comes from the father. It is therefore a non-molar miscarriage.
Figure 9: Representative genotyping results of patient 1790. (A) Select genotyping results of the patient's first conception showing a triploid dispermic genotype. (B) Select genotyping results from the patient's second conception showing a triploid digynic genotype. The x-axis is in basepairs; the labels and basepair sizes have been omitted for simplicity. The y-axis represents peak height and is similarly omitted from the figure for simplicity. Please click here to view a larger version of this figure.
This patient was initially misdiagnosed with two PHM and was worrying about an increased risk of more moles while she had a single PHM. This case highlights the limitation of SNP microarray on the POC alone. SNP microarray is a powerful method and is the best to detect aneuploidies of any chromosome (trisomies, monosomies, or non-diploid genotypes); however, when performed on the POC alone without analyzing parental DNA, the origin of the triploidy cannot be determined. For further explanations about genotype analysis and interpretation, please refer to the study by Murphy et al.16.
Representative results shown in Figure 10A are of a triploid conception. The values of the x-axis represent the nuclear DNA content. For example, 200 is an arbitrary number given to cells that contain a certain amount of nuclear DNA content. Therefore, a peak at 400 represents cells that contain double the amount of nuclear DNA content as compared to the 200 peak. The little peak around 300 represents nuclear DNA content that is in between the 200 and 400 peak and is therefore the triploid peak. Notice how a diploid conception (Figure 10B) does not contain any peak at the 300 value.
In some cases, the triploid peak is very subtle (Figure 10C). Whenever a barely noticeable triploid peak is noted, it is important to first consider the amount of CV that were present in the sections used for the flow cytometry analysis. If the sections had less than about 20% CV, then it will likely be a true triploidy since it is expected to be a very low peak. If the sections taken had high amounts of CV, the POC becomes suspicious of mosaicism with the presence of another diploid cellular population. This can be checked by re-reviewing the genotyping results to see if they fit a perfect triploid dispermy or can also be confirmed by FISH with probes from the X, Y, and 18 chromosomes. Also, using the software described in this article, it is possible to set specific gates, as described in section 2.4.1, that allow the user to focus on a specific region to enrich for triploid cells if they really exist.
Figure 10: Representative flow cytometry results demonstrating triploid conceptions in (A) and (C), and a diploid conception in (B). Please click here to view a larger version of this figure.
HM are abnormal human pregnancies with heterogeneous etiologies and have different histological and genotypic types, which makes their accurate classification and diagnosis challenging. Histopathological morphological evaluation was often proven inaccurate and is therefore unreliable on its own to classify HM into CHM and PHM and distinguish them from non-molar miscarriages. Therefore, an accurate diagnosis of HM requires the use of other methods such as multiplex microsatellite DNA genotyping, ploidy analysis by flow cytometry, ploidy analysis by FISH, and p57KIP2 immunohistochemistry. Each of these methods has its own limitations and advantages.
Limitations and advantages of multiplex genotyping and flow cytometry
Maternal contamination is one of the most common issues when working with FFPE tissues and may lead to misdiagnosis, thus highlighting the importance of separating maternal from POC tissues. It is important to identify maternal contamination in order to have an idea of what to expect of the genotyping peaks and to help with their interpretation. If the level of contamination is too large and prevents reliable interpretation of the results, then repeat the DNA isolation and extraction, taking extra care in removing all possible maternal tissues. In regions where the morphology of the tissues is not clear, it is better to remove such regions to minimize the chance of maternal contamination. The first advantage of the method described in this protocol, in addition to its lower cost, is that maternal tissues are removed from the slides while other methods consist of collecting POC tissues (by covering them with solution that polymerizes when exposed to air and lifting the tissues) without removing maternal tissues. The method described here thus allows one to take a second look at the remaining POC tissues, re-clean them if necessary, as is often the case, and then collect and extract DNA from them. The second advantage is that we clean tissues on uncoverslipped H&E stained sections, which greatly facilitates the removal of maternal tissues, as opposed to the use of unstained sections and a coverslipped map slide. However, the additional staining may further degrade the DNA, and this is compensated for by adding more sections.
The availability of parental DNA for the analysis is another challenge. The presence of both parents greatly facilitates the analysis and interpretation of genotyping results. Unfortunately, however, it is quite often the case that the father's DNA is not available, which may sometimes complicate the analysis, especially in cases where the quality of the POC DNA is poor due to prior fixation or long-term storage (more frequent when working with recurrent HM). Furthermore, maternal blood may not always be available for DNA extraction. In such cases, maternal DNA can be extracted from maternal endometrial tissues present in the FFPE blocks as previously described16. Also, characterizing molar tissues that resulted from the use of assisted reproductive technologies may complicate microsatellite genotype analysis because, in most of the cases, DNA from the donors (males or females) is usually not available.
Lastly, the fact that larger peaks tend to be shorter in terms of peak height is another possible source of confusion, particularly when the peak heights need to be used to determine if there are two doses or one dose in a single peak. One way to overcome this is to always keep in mind that peaks of large allele size (number of base pairs) tend to be shorter, due to the degraded nature of DNA from FFPE tissues, which makes less DNA available for the amplification of larger alleles. In addition, a shorter PCR fragment amplification takes less time than a larger one, and the exponential amplification of DNA leads to fewer amounts of larger alleles.
The main drawback of flow cytometry is that triploid conceptions may sometimes be missed, and this could be due to insufficient amounts of CV in the block. However, the presence of a triploid peak is a conclusive indication of a triploidy. Note that this method is not sensitive enough to detect trisomies, tetraploid conceptions, or other aneuploidies using this protocol. Tetraploid conceptions are not detectable by this protocol because the tetraploid peak corresponds to the same peak of diploid cells in the G2 phase of the cell cycle.
Knowing these limitations and challenges helps in reducing mistakes. It is thus important and sometimes necessary to analyze the same tissue with different methods, compare the results, and make sure that they are concordant with one another. If they are not, results need to be reconsidered and analyses need to be repeated. For the several cases that showed conflicting results, discrepancies were resolved simply by repeating the experiments and taking appropriate care to avoid the original problem. In other cases, discrepancies were resolved by performing additional methods such as FISH on tissue sections or by performing additional simplex genotyping with appropriate markers.
Identification of maternal contamination
With respect to identifying contamination of the POC DNA with maternal DNA, the level of contamination will be reflected or recognized by the presence of all maternal alleles at all loci in the POC, in addition to the maternal allele(s) that are transmitted to the POC.
In Figure 11A, peaks originating from maternal DNA contamination are labelled with a "c". Notice how every peak marked with a "c" is also present in the mother, and we see these "c" peaks in all three markers. Note that for the second marker (in blue), the maternal contamination peak indicated by a "c" is higher than each "c" peak at the first marker because the mother is homozygous for the second marker; the height of the contaminant is therefore doubled for this marker. In this POC, there are no maternally derived peaks. In other words, this POC did not inherit any alleles from the mother. There is only one real peak at each marker and this peak is not present in the mother. We therefore know that the real peaks must have come from the father. With ploidy information from either karyotype or flow cytometry analysis demonstrating diploidy, it is possible to conclude that this POC is both androgenetic in origin and diploid.
Furthermore, these three markers in Figure 11A reveal that there is always only one real peak for every marker. Only three markers are illustrated here, however, multiplex kits often come with many more markers that will also reveal the same pattern. Since this POC is diploid, there must be two doses in each peak. With this information, we can conclude that this POC is androgenetic monospermic and is homozygous at every single marker.
Figure 11B represents a diploid biparental POC. The little black bar across the first peak of the first marker (in green) represents the estimated amount of contamination that is present within this real peak. The "R" indicates the real portion of this peak coming from the POC DNA; the "c" represents the contaminant portion of this peak coming from the maternal DNA. How can one identify the level of contamination? It is possible, in this case, when a marker is heterozygous in the mother because one of the maternal alleles is absent in the POC. The small peak in the first marker labelled with "c," for example, indicates that this is the level of contamination that should be expected for the other maternally inherited peak. Consequently, all the POC peaks that are of maternal origin are slightly higher than the paternally inherited peaks due to the small added amount of contamination. For the third marker (in black) in Figure 11B, the level of contamination is expected to be double the usual amount (because the mother is homozygous for this specific marker), and one can therefore infer that there is only one dose in the first peak in the POC.
Figure 11C illustrates a triploid dispermic POC. The first marker (in green) shows three peaks in the POC. Since these three peaks have similar heights, it is possible to conclude that this POC is likely triploid. Note that the third peak in this marker is shorter than the first. This is expected because, as a general trend, larger peaks tend to be shorter. The second peak is slightly higher than the first, and this can be accounted for by a small amount of maternal contamination, as indicated by the bar and "c." Furthermore, two of these three peaks are not present in the mother, and therefore must have come from the father. Thus far, this marker indicates that the POC could be triploid (or a trisomy) and that the origin of the extra set of chromosomes is paternal. It also indicates that it is a dispermic conception, since the POC inherited two different alleles from the father.
The second marker (in blue) in Figure 11C has only two peaks. After accounting for the level of contamination, the second peak appears higher than the first, and this is despite the general trend for larger peaks to be smaller (a trend that must always be kept in mind during analysis). Thus, it is likely that there are two doses in that one big peak. This is also supported by the fact that the first marker is indicative of triploidy.
Lastly, the third marker (in black) in Figure 11C shows three peaks, again indicative of triploidy. Since most markers in multiplex kits come from different chromosomes, it is possible to conclude with confidence that a POC is triploid after observing the same trend of three alleles across several different markers. Also note from this marker that two out of the three peaks originate from the father, confirming the dispermic origin.
Figure 11: Genotyping results illustrating the effect of maternal contamination. The top panels show the alleles that belong to the POC and the bottom panels show those that belong to the mother. In (A), the POC is androgenetic monospermic and the level of contamination is highlighted by an arrow and the letter "c." In (B), the POC is diploid biparental, and the level of contamination is highlighted by the letter "c." In (C), the POC is triploid dispermic and the level of contamination is highlighted by the letter "c." The little black bars show how much of the peak heights comes from maternal contamination and should therefore be taken into consideration when comparing peak heights. The x-axis is in basepairs; the labels and basepair sizes have been omitted for simplicity. The y-axis represents peak height and is similarly omitted from the figure for simplicity. Please click here to view a larger version of this figure.
Examples of challenging cases
For a number of cases, it was only possible to arrive at a conclusion by having all three assessments simultaneously (i.e., flow, p57KIP2, and multiplex genotyping). For example, one case (808) was referred as a non-molar miscarriage. Histopathological evaluation lead to suspicion of a molar conception and genotyping analysis of the POC revealed one marker that clearly showed three peaks (two from the father and one from the mother). The p57KIP2 results were not conclusive. The flow cytometry analysis revealed a small triploid peak that was confirmed with FISH analysis, which also ruled out the presence of another diploid cellular population. With confirmation from FISH, it was possible to conclude that the POC is indeed a triploid dispermic mole.
Another case (1192) was also referred as non-molar miscarriage. The CV of this POC were intermingled with maternal tissues and were highly necrotic as well, making the cleaning process very challenging. Consequently, the first genotyping analysis showed a high level of maternal contamination, such that every maternal allele could be seen in the POC (a good indication of possible contamination). Furthermore, the p57KIP2 was unfortunately inconclusive, most likely due to the necrotic nature of the tissue, perhaps because of delayed fixation. The flow cytometry results, however, were indicative of triploidy, which was also confirmed by FISH. The DNA was re-extracted with the goal of reducing the level of contamination and removing tissues with unclear morphology. Analysis of the new genotyping results, while accounting for the probable presence of maternal contamination, allowed us to conclude that the POC was a triploid dispermic XXY PHM.
Table 4 outlines the typical methods used for analysis. It is recommended to use morphological evaluation and p57KIP2 immunohistochemistry on all tissues suspicious of HM and at least one genotyping method. Among genotyping methods, the most informative is multiplex DNA genotyping. When results between different methods are not concordant or when some results are inconclusive, other genotyping methods need to be used. The table demonstrates expected concordant results for each possible HM type along with rare exceptions and recommendations to solve them.
<!–Morphology compatible with: |
CHM | PHM | Twins (CHM + fetus)/ Mosaic |
P57 IHC | Negative | Positive | Positive and negative |
Multiplex microsatellite genotyping of POC and mother | Diploid Androgenetic Monospermic – 85% Diploid Androgenetic Dispermic – 15% |
Triploid Dispermic – 99% Triploid Monospermic – 1% |
Mosaic: Dip And + Dip Bip 1 – One non-maternal allele – monozygotic 2 – Two non-maternal alleles – dizygotic Recommendation: – For mosaic cases, FISH with X, Y, and 18 may help. – Compare FISH with p57. – Flow cytometry to exclude triploid cells. |
Discrepancies and exceptions: |
Diploid Biparental Morphology: CHM or not typical CHM/PHM. p57 -, p57+/-, or p57+. Recommendation: Re-review reproductive history, analyze other POCs, and search for mutations in NLPR7/KHDC3L/PADI6. |
||
1 – Dip And and p57+ retained maternal Chr 11/11p15 Recommendation: Simplex Chr 11 markers, FISH. 2 – Dip Bip and p57 – deleted maternal Chr 11/11p15 Morphology: Not typical CHM, no TP Recommendation: Simplex Chr 11 markers, FISH. |
1 – Dip Bip aneuploid MC, up to 40% Morphology: Not typical PHM, no/mild TP Recommendation: Microarray 2 – Triploid Digynic Morphology: Not typical PHM, p57 ++ 3 – Tetraploid (Trispermic or 2 mat and 2 pat sets of Chr.): <1% Morphology: Not typical PHM Recommendation: FISH |
Table 4: Typical order of analysis, expected results, and rare exceptions along with recommendations to solve them. P57KIP2 immunohistochemistry aims to detect the expression of p57KIP2, the protein coded by CDKN1C gene. This gene is paternally imprinted in the cytotrophoblast and the villous stroma of first trimester placenta and is expressed only from the maternal genome. It is therefore used as an ancillary marker to detect, in an easy and inexpensive way, the presence of the maternal genome in the POC. It is recommended to perform p57KIP2 immunohistochemistry for all POCs suspected to be HM in parallel to the morphological evaluation. In the author's laboratory, the p57KIP2 antibody and platforms indicated in the Table of Materials are used and the authors are very happy with the quality of the results. Abbreviations: IHC = immunohistochemistry; Dip And = diploid androgenetic; Dip Bip = diploid biparental; Chr = chromosome; MC = miscarriage; and TP = trophoblastic proliferation.
To the best of the authors' knowledge, this article is the first to provide detailed protocols for flow cytometry as well as low-cost and high-quality multiplex microsatellite DNA genotyping of FFPE POC tissues. The interpretation of the results is also described, along with their troubleshooting and integration with those of other methods to reach accurate conclusions and diagnoses of POCs and HM. The authors sincerely hope that this article can be helpful for researchers trying to understand this complex entity.
The authors have nothing to disclose.
The authors thank Sophie Patrier and Marianne Parésy for sharing the original flow cytometry protocol, and Promega and Qiagen for providing supplies and reagents. This work was supported by the Réseau Québécois en Reproduction and the Canadian Institute for Health Research (MOP-130364) to R.S.
BD FACS Canto II | BD BioSciences | 338960 | |
Capillary electrophoresis instrument: Genomes Applied Biosystems 3730xl DNA Analyzer | Applied biosystems | 313001R | Service offered by the Centre for Applied Genomics (http://www.tcag.ca) |
Citric acid | Sigma | 251275 | |
Cytoseal 60, histopathology mounting medium | Fisher | 23244257 | |
Eosin Y stock solution (1%) | Fisher | SE23-500D | |
FCSalyzer – flow cytometry analysis software | SourceForge | – | https://sourceforge.net/projects/fcsalyzer/ |
FFPE Qiagen kit | Qiagen | 80234 | |
Forceps | Fine Science Tools | 11295-51 | For sectioning and for the cleaning process |
Glacial Acetic Acid (Concentrated) | Sigma | A6283-500mL | |
Glass coverslips: Cover Glass | Fisher | 12-541a | |
Hematoxylin | Fisher | CS401-1D | |
Highly deionized formamide: Hi-Di Formamide | Thermofisher | 4311320 | |
IHC platform: Benchmark Ultra | Roche | – | |
Kimwipes | Ultident | 30-34120 | |
Microtome | Leica | RM2135 | |
Microtome blades | Fisher | 12-634-1C | |
Nitex filtering mesh, 48 microns | Filmar | 74011 | http://www.filmar.qc.ca/index.php?filet=produits&id=51&lang=en ; any other filter is suitable, but this is an inexpensive and effective option from a non-research company |
p57 antibody | Cell Marque | 457M | |
Pasteur pipette | VWR | 53499-632 | |
PCR machine | Perkin Elmer, Applied Biosystems | GeneAmp PCR System 9700 | |
PeakScanner 1.0 | Applied Biosystems | 4381867 | Software for genotyping analysis. |
Pepsin from porcine gastric mucosa | Sigma | P7012 | |
Polystyrene round-bottom tubes | BD Falcon | 352058 | |
Positively charged slides: Superfrost Plus 25x75mm | Fisher | 1255015 | |
PowerPlex 16 HS System | Promega Corporation | DC2102 | |
Propidium Iodide | Sigma | P4864 | |
Ribonuclease A from bovine pancreas | Sigma | R4875 | |
Separation matrix: POP-7 Polymer | Thermofisher | 4352759 | |
UltraPure Agarose | Fisher | 16500-500 | |
Xylene | Fisher | X3P1GAL |