US Biomax, Inc.

FAQs

Tissue Microarray FAQs

Telomerase Detection Kits

  1. How long do you keep tissue out before you put them in fixative?
  2. How long do you fix your samples? What fixative do you use?
  3. How long do you fix your samples? What fixative do you use?
  4. What kind of decalcification method do you use?
  5. Do you offer fixed tissue microarrays? How many samples can you put on one array?
  6. How are slides processed?
  7. What is the post-mortem interval/time to fixation for the autopsy samples?
  8. How long is there between tissue collection and fixation for surgical cases?
  9. Is the tissues used in making tissue arrays validated for immunohistochemistry studies?
  10. What ethical considerations and protocols are used in tissue collection?
  11. Do we need to have a Material Transfer Agreement (MTA) in place to order from US Biomax?
  12. How long can a tissue array slide can be stored?
  13. Are tissue microarrays valid for clinico-pathological studies? In other words, whether tissue arrays account for heterogeneity of tissues or do ONE small core in tissue array representative of a whole tissue section?
  14. How can you make sure the tissue array cores are pathologically represented the original tissue block?
  15. What's the threshold of your tissue array standard?
  16. We are still not convinced; can we try your tissue microarray before purchasing?
  17. Do you have protocols you recommend to use for IHC or ISH on your tissue arrays?
  18. What's the minimum amount of tissue arrays for one order?
  19. What's the maximum amount of tissue arrays we can buy?
  20. How do custom arrays work?
  21. Do you use adhesive tape for transferring tissue sections on your tissue microarray (gene-chip)?
  22. What's the advantage of your microarray over other vendors?
  23. Why do you use www.biomax.us as your company's domain?

Q: How long do you keep tissue out before you put them in fixative?

Samples are typically put into formalin within 15 - 30 minutes after surgical resection. Some of the tissue samples are snap frozen in liquid nitrogen, which provides better preservation of tissue morphology and architecture than that of in OCT.

Q: How long do you fix your samples? What fixative do you use?

Surgically removed samples were preserved in neutral phosphate buffered formalin for 24 hours before they are processed in a tissue processor (Leica) and embedded in paraffin.

Q: Do you offer frozen tissue microarrays? How many samples can you put on one array?

Unfortunately, we provide pre-made frozen tissues arrays in OCT-blocks for standard cryosections. We can also make custom frozen tissue microarrays. These frozen tissue array sections are suitable for immunohistochemistry staining and in situ hybridization studies. Please contact us for details.

Q: Do you offer fixed tissue microarrays? How many samples can you put on one array?

Yes, we make formalin-fixed tissues embedded and arrayed in paraffin for high throughput immunohistochemistry staining and in situ hybridization. We can put ~80 cores with a 1.5 mm diameter, ~200 cores with a 1.0 mm diameter, or ~800 cores with a 0.6 mm diameter on one tissue array.

Q: What kind of decalcification method do you use?

There is standard 24-48 hour 10% nitric acid treatment (decalcification) after 24 hour of fixation in neutral buffered formalin.

Q: How are slides processed?

All tissues are fixed in 10% buffered PBS neutral formalin for 24 hours, dehydrated with gradient ethanol, cleared with xylene, and embedded in paraffin. Afterwards, each slide is tested for immunohistochemistry on one antibody specific to the tissue used in the array. Quality control is ensured as every tissue block is collected and arranged by our pathologists.

Q: What is the post-mortem interval/time to fixation for autopsy samples?

For post mortem tissues, the typical time in formalin fixative depends on the duration of procedures performed after death, including the acquisition of consent from the family. Nevertheless, the post mortem interval is less than six hours.

Q: How long is there between tissue collection and fixation for surgical cases?

After surgery, tissue samples are put into formalin and brought to the pathology department of our collaborating hospitals. This process occurs less than 10 minutes after surgery and before the fixation.

Q: Is the tissues used in making tissue arrays validated for immunohistochemistry studies?

Yes, two tissue sections of every lot of tissue arrays was sampled for IHC studies to ensure the validation of antigenicity remains in the tissue and combined with H&E slides of every 10th sections to assure the tissue were properly fixed and processed.

Q: What ethical considerations and protocols are used in tissue collection?

All tissue is collected under the highest ethical standards with the donor being informed completely and with their consent. We make sure we follow standard medical care and protect the donors' privacy.

All human tissues are collected under HIPPA approved protocols. All animal tissues are collected under IACUC protocol. All samples have been tested negative for HIV and Hepatitis B or their counterparts in animals, and approved for commercial product development.

Q: Do we need to have a Material Transfer Agreement (MTA) in place to order from US Biomax?

It is not required to have an MTA signed for research orientated institutions, as well as pharmaceutical companies who need to produce therapeutic and diagnostic reagents using our tissue samples or tissue arrays. It is the individual researcher's responsibility to get approval from their respective Intuitional Review Board (IRB) to use human tissue samples for their research work. We can provide necessary documents to satisfy your needs in order to get approval from your IRB

Q: How long can a tissue array slide can be stored?

After sectioning, tissue slides from US Biomax were baked at 60°C for 2 hours. We have tested that the antigenicity was not lost (no significant difference in statistics) with a tissue array slide kept in a refrigerator (4°C) dated 4 years ago. We have found that simplicity is the best — there is no need to have a thick layer of paraffin or a fancy nitrogen bag.

Q: Are tissue microarrays valid for clinico-pathological studies? In other words, do tissue arrays account for heterogeneity of tissues or does ONE small core in a tissue array represent a whole tissue section?

Several studies have examined the question of the representation of tissues in TMAs using various markers in different tumor types. In general, although they vary somewhat in terms of recommendations for sampling, all studies indicate there is usually excellent agreement between the use of TMAs and standard tissue sections for clinico-pathological studies.1 Their conclusion is that for homogeneous markers, a single TMA spot (0.6 mm) per case will be adequate.

Q: How can you make sure the tissue array cores are pathologically represented the original tissue block?

Tissue arrays from US Biomax usually consist of cores from 1.0 to 1.5 mm in diameter, which is an area 2.7 to 6.3 times larger than that of a 0.6 mm core tissue microarray. Thus, 1.0 mm and above core size is sufficient to represent the whole tissue section. Our high-density tissue arrays use high numbers of single cores to maximize the numbers of tissue to benefit the end users.
Every 10th section of the tissue array is stained with H&E and reviewed by two well experienced pathologists to make sure the pathology diagnosis is current and matched to the adjacent serial sections.

Q: What's the threshold of your tissue array standard?

US Biomax is the first company to adopt the highest quality control standard in making tissue arrays, which is 90% valid cores remaining in a given tissue array. A valid core means that the tissue core not only remains 50% or higher intact, but its pathology is also confirmed by an experienced pathologist. In other words, less than 10% of cores fall off or become invalid (e.g. cancer becomes normal). In reality, all of our tissue arrays meet a 95% valid core QC standard.

Q: We are still not convinced; can we try your tissue microarray before purchasing?

We have limited numbers of trial slides (QC standard not met, but at least 70% of cores remain; good for titrating antibody and retrieving conditions) for certain arrays at $15 - $85 each (1/4 of regular price), depending on the amount of cores. To add a trial tissue array slide is easy. Just click the adjacent price link to the right of our regular tissue array slide in our online shopping cart. You may only purchase up to two trial slides at a time. The catalog number is trial slide is regular catalog number with t at the end.

Q: Do you have protocols you recommend to use for IHC or ISH on your tissue arrays?

Yes, we do. You can download the protocols in PDF format under our support page.

Q: What's the minimum amount of tissue arrays for one order?

One unstained paraffin tissue array slide.

Q: What's the maximum amount of tissue arrays we can buy?

As many as you want. The more you purchase, the cheaper in single unit price. Please contact us for details.

Q: How do custom arrays work?

Option 1: you provide tissue blocks, and we assemble the tissue array for you. Contact us to get a detailed quote.
Option 2: we provide tissue blocks from our tissue bank for your tissue array. There is a minimum number of 10 tissue sections to purchase at regular price with similar numbers of cores of our pre made arrays, and there is NO setup fee or other hidden cost. You do not need to purchase the whole block, which is also option at 50% discount of regular single slide price. Our regular tissue microarray block usually can produce 200-350 good sections.

Q: Do you use adhesive tape for transferring tissue sections on your tissue microarray (gene-chip)?

No more messy transferring steps and expensive tapes for us in making tissue arrays. The amount of adhesive material remaining in slides can possibly interfere with the subsequent immunohistochemistry staining or in situ hybridization procedures. Our tissue arrays are sectioned and mounted manually.

Q: What's the advantage of your microarray over other vendors?

Every vendor of tissue arrays has its own niche, either by providing varieties of tissue types or numbers of core format to meet needs for end users. The advantage of tissue arrays in US Biomax include:
  • Tissue blocks for making tissue array which were recently collected within 5 years, for making tissue microarray in mind.
  • High quality, stringent QC standard (90% to 95% valid cores remain in a given tissue array).
  • Strong adherence of tissue array sections with glass slides to sustain the rigorous antigen retrieving procedures (heat, microwave oven or boiling, or high and low pH). Tissue array sections are mounted on Fisher SuperFrost Positive Charged glass, the highest quality available in the industry.
  • Fresh, paraffin coated thin layer on tissue microarray sections to prevent oxidation or moisture condensation which may lead to enzyme digestion or mold growing.
  • Low price specificity (price/slide) while providing high number of cases and larger core areas. For example, PR802, priced at $120 per slide, consists of 78 distinguished cases of prostate cancer, 2 cases of normal prostate tissue, while maintaining 1.5 mm diameter cores. Other examples include: CO2001, LV2001, BR2001, and LC2001, which have 192 cases of cancer and 8 cores of normal tissue with 1.0 mm diameter core, priced at $295 each tissue array. The other high density TMAs, such as MC5001 with 500 cores, 0.6 mm core size or BC01081 with 616 cores, 0.6 mm core size are good example of our techniques to build high density tissue microarrays.
  • Varieties of disease types available. We provide more than 320 types of tissue arrays, and 30 or so types of diseased or normal tissue arrays to choose from. Some rare types of cancer are also in our inventory, such as adrenal carcinoma, penile, vulvar, brain glioma, lymphoma, pancreatic cancer, sarcoma, chondrosarcoma, melanoma, head and neck tumor tissue arrays, besides common types of cancer arrays (such as cervical, colon, prostate, breast, lung, liver, kidney, thyroid, esophagus, pharyngeal, laryngeal and skin).
  • Maximum normal controls available for most types of cancer. For instance, BR803 contains 40 cases of breast cancer and 40 cores of matched adjacent normal tissue from the same donor, while maintaining a 1.5 mm core diameter. Another example is BR1001, containing 50 cases of breast cancer and their matched metastasis in lymph nodes.
  • We ship our tissue arrays with FedEx standard overnight delivery to all 50 states in the U.S. and International Priority to most other countries. Orders received before 4:00pm EST will be sent out in the same day. We only list items in stock, although you may backorder from us.
  • All tissue arrays are listed with price and package information. You can choose to buy trial (regular catalog number ending with t) and H & E stained slide (regular catalog number ending with s) as well as with regular tissue arrays. Best of all, you can place orders online with a Purchase Order or a Credit Card, all without discount. It's safe, convenient and fast.

Q: Why do you use www.biomax.us as your company's domain?

Our company is called US Biomax, so we chose the reciprocal name biomax.us for our domain, which is a perfect match: short, concise and fitting to our name. We also own www.usbiomax.com, which redirects here. By the way, we do not have any connection with Biomax GmbH (Bioinformatics company) in Germany, nor with Biomax Inc. in Baltimore who sells spin column.

Telomerase Detection Kits

Q: How many samples can be analyzed per kit?

Single reaction:
MT3010: 50 - 7(TSR) - 1(Buffer) = 42 / 3 dilutions = 14 samples
MT3011: 100 - 7(TSR) - 1(Buffer) = 92 / 3 dilutions = 30 samples
MT3012: 200 - 7(TSR) - 1(Buffer) = 192 / 3 dilutions = 64 samples

Duplicate:
MT3010: 50 ? 7x2 - 1x2 = 34 / 3 / 2 = 5 samples
MT3011: 100 ? 7x2 - 1x2 = 84 / 3 / 2 = 14 samples
MT3012: 200 ? 7x2 - 1x2 = 184 / 3 / 2 = 30 samples

Q: How much protein is needed for each assay?

The working range for 293T cell extract or equivalent is 0.5 ng-1000ng per PCR reaction. For other proteins, use no more than 1mg per reaction.

Q: How many dilutions are needed for each sample?

We recommend that you do at least 3 serial dilutions for each sample.

Q: Do you recommend double-testing the standard curve?

It is not necessary to double-test the standard curve due to its sufficient stability; however, if desired it can be done at the beginning of the experiment until the user is comfortable with the assay.

Q: For how long do you incubate the cells with the lysis buffer and at what temperature?

For 6-well plate:

  • Wash cell with 2 ml PBS per well, RT. Add 0.5 ml Trypsin to each well.
  • Incubate 5min at 37°C. Add 3 ml cell culture medium to each well and harvest to 15 ml tube.
  • Centrifuge at 1000 x rpm, 5min at 4°C. Remove supernatant and add 3 ml cold PBS washing.
  • Centrifuge at 1000 x rpm, 5min, 4°C. Remove PBS.
  • Add 100~200µl of 1 x Lysis Buffer per 105-106 cells, mix well and incubate for 30min at 4°C. Centrifuge, 13,000 x rpm, 30min at 4°C.
  • Transfer the supernatant for analysis.

Q: Is the 1`Lysis Buffer compatible with BCA protein assay reagent?

Yes. You also can use RC DC kit from Bio-Rad to measure the protein concentration. There are no components in the buffer that will interfere with the measurement.

Q: What can we do if the provided lysis Buffer is not enough?

We sell the 1xLysis Buffer separately. Catalog # MT3002. Or you can stay with the lysis buffer you are currently using to prepare your samples, and then use the provided buffer to make the dilutions.

Q: Which fluorescent should be selected on our real-time PCR machine?

Select FAM-490 on the PCR machines from most vendors, such as Bio-Rad, Stratagene. For others, please consult the manufacture for specifications.

Q: What is the purpose of the ?melting curve? step and when should it be performed?

The melting curve step is to verify the content of PCR product. The melting point is the denature temperature of double-stranded DNA. The longer the DNA fragments, the higher the melting temperature. Primers may form dimmers and their melting points are different from the amplified DNA fragments.

Q: How stable is the QTD kit?

It is quite stable (up to several days at room temperature).

Q: How long is the shelf life of the QTD kit?

For at least 1 year at 20°C.

Q: Can PCR products be analyzed by PAGE?

No.

Q: I expect that my cells express very low levels of telomerase activity. Can I try to make a more "concentrated" sample by using less lysis buffer?

Yes.

Q: Can the result be quantified by comparing with TSR standard?

TSR standard Ct value gives a reference for quantification.

Q: I was not able to reach the same standard curve of TS R8 as appears in the protocol and also the results were not reproducible. In every test I got a different CT.

Try to adjust the threshold baseline to be the same for every test.

Q: The positive control just didn°št work of using your Telomerase Detection Kit on Roche LightCycler. We tested on conditions of your mannual or Roche conditions.

The Roche lightcycler has a BSA problem. The customer need to add BSA (BSA may be required if the reaction is run on a Roche. LightCycler. A final BSA concentration of 0.5mg/mL may be sufficient.) to the mixture to resolve the problem if they prefer to use lightcycler.

Please see the links to have more information.
http://www.etendx.com/support/EvaGreen_20X.pdf
http://www.wistar.org/research_facilities/facilities/genomics/realtime_qPCR.pdf
http://www.bio.net/bionet/mm/methods/1999-September/078069.html
http://www.bio.net/bionet/mm/methods/2000-October/085668.html

Antibody Microarray

Q: What are the major applications of the antibody microarray?

The antibody arrays are designed for qualitative profiling of protein expression, screening, and comparison between normal, diseased or treated samples.

Q: How many slides are included in one set of arrays?

There are two slides included in each set of arrays. There is one array on each slide for analyzing one sample. You can analyze two samples with each set of arrays.

Q: What samples can be used for analysis and detection?

Proteins from cell extracts, tissue lysates, or serum samples can be used for analysis.

Q: Do I need to run a control/known sample?

It is recommended to run a control or known sample along with the test samples.

Q: What does it mean when an antibody has a name like this, p53(ab-15) or p53(phospho-Ser15)?

The number in parenthesis in an antibody's name indicates the site of phosphorylation . For example, p53(Ab-15) was made from a synthetic peptide (non-phosphorylated) derived from human p53 around the phosphorylation site of Serine 15. It detects endogenous levels of total p53 protein. p53(phospho-Ser15) means that the antibody was derived from a synthetic phosphopeptide derived from human p53 around the phosphorylation site of Serine 315. It detects endogenous levels of p53 only when phosphorylated at Serine 315. Both phospho-specific antibodies and their non-phospho pairs are included in the array.

Q: In the phospho-antibody arrays, why are there multiple antibodies for a single protein?

They are highly specific antibodies made to recognize the different phosphorylation sites on the same protein. For example, c-Jun(Phospho-Thr91) detects endogenous levels of c-Jun only when phosphorylated at Threonine 91; c-Jun(Phospho-Tyr170) detected endogenous levels of c-Jun only when phosphorylated at Tyrosine 170.

Q. What are the key steps in getting good results?

The first key step in getting good results is protein extraction. First, make sure to vortex the beads with cells for 30 seconds every 10 minutes for 1 hours. This process will fully disrupt the cells/tissues and release protein. Secondly, it is very important that the cell lysate supernatant is very clear. Impurity in the supernatant ( dirty lysate ) can cause low labeling efficiency and non-specific binding. Be sure to only use the top, clear layer of the lysate supernatant for labeling. The supernatant should be as clear and transparent as water. If the supernatant still appears cloudy (unclear) at the end of the extraction protocol, freeze it at -70 for 10min, then spin at 10000xg. Save the clear layer of the supernatant. Another important step is the rinses with DI water. It's absolutely crucial to wash the slides EXTENSIVELY with DI water after blocking, coupling and detection. Increase the number of washes if needed. Agitate the water during wash. It will help remove any residual reagent from the slide surface. Here is a short video showing how we wash the slides with DI water. http://www.youtube.com/watch?v=mst7vnahpP8 If you don't use slide racks, you can submerge the slide under water and then move it back and forth.

Q. How many cells are needed to obtain 100 micrograms of protein?
The amount of protein present in cells may vary with cell type. We typically use 1 to 10 million cells to get approximately 1mg of protein; only 10uL of which containing less than 100ug of protein is used for labeling. Start with 5 million cells whenever possible.

Q: Can I use other types of cell lysis and/or extraction buffer instead of the Extraction buffer provided in the Array Assay Kit?

Yes, it is possible to use other lysis buffers to lyse cells; however, the buffer must be free of Tris. The presence of Tris in cell lysates or extracted protein sample can adversely affect biotinylation of protein samples. For instance, the RIPA Lysis and Extraction buffer from Pierce Biotechnology contains Tris. If this buffer was used to extract proteins from cells, please be sure to remove the buffer from your protein extract and replace with the Labeling Buffer provided in the Antibody Array Assay Kit before proceeding to the next step. We recommend the following columns for buffer exchange (removing Tris): Spin columns included in the Antibody Array Assay Kit; Millipore, Microcon YM-10 filters (Catalog: 42406); Sephadex G-25 columns.

Q: Can I add protease inhibitors to the lysates ?

The reagents provided in the Antibody Array Assay Kit do not contain protease inhibitors. To prevent protein degradation, once you start the extraction, you should work quickly and proceed diligently towards the array analysis step. Alternatively, you may use inhibitors if you prefer or plan to store the proteins for a week or longer.

Q. What is the minimum amount of proteins needed for coupling?

Typically 40-100ug of total protein is used for biotin labeling and coupling.

Q. Which method is better for drying the slide?

For drying the slide after detection, the goal is to remove water from surface as quickly as possible. Compressed nitrogen works better than centrifugation. It's faster and more efficient. Before using nitrogen, hold the slide with your fingers and shake off excess water as much as you can. Then, point the nitrogen nozzle at a 30 degree angle about 20mm away from the slide surface, use the air to push the water off the surface quickly without touching the slide surface. Be sure to keep the air pressure between 20-30psi.

Q: Do I have to use Cy3-Streptavidin for detection?

No, you do not have to use Cy3-Streptavidin. As alternatives, you can use Cy5-Streptavidin, or Alexa Fluor 532 or 647 conjugated streptavidin.

Q: How important is it to rinse the slides extensively with DI water after the slides are subject to blocking buffer and/or wash buffer?

It is extremely important to rinse the slide extensively with DI water. Any residual reagents on the slide surface may cause non-uniform background. Rinsing the slides with DI water extensively helps achieve better background uniformity.

Q: How long can the finished arrays be stored before they are read on a scanner?

The arrays should be read (scanned) as soon as possible after the experiment is complete. If you do not have access to a scanner immediately, store the finished arrays in a non-transparent box (to protect them from light) at room temperature for two or three days before they can be read on a scanner.

Q: What scanners can be used for detection?

Any fluorescence based scanner, that is compatible with 3 in x 1 in (76 mm x 25 mm) microscope slides, can be used for detection. Click here for a list of compatible and incompatible systems. If you do not have access to a scanner, we will be happy to scan the arrays for you and provide raw data in Excel format.

Q: Do you provide a software for image analysis?

We do not provide an image analysis software. Any image analysis software that came with the scanners or any type of spot finder software can be used for image analysis. We do provide a GAL file (GenePix Array List) for each array, which can be used to set up grids for image analysis. You can find more information about GAL files at http://www.moleculardevices.com/pages/software/gn_genepix_file_formats.html#gal.

References

  1. A.M. DeMarzo, H. Fedor, "The principles, uses and construction of tissue microarrays in pathology research," Presentation at the 92nd Annual Meeting of the United States and Canadian Academy of Pathology, March 27, 2003, Washington DC.