Sample Preparation and Shipping

For H&E, Special Stain (SS), IHC, IF, or ISH staining, we recommend the following protocol:

Note: For ISH testing, contact client support for specific protocols.

1. Fix your tissue in 10% Neutral Buffered
2. Formalin (10% NBF) or 4% PFA for 24–36 hours at room temperature. Please note that tissue thickness should be between 3-5 mm for optimal penetration.
3. After fixation is complete, transfer the tissue to 70% ethanol and ship overnight for FFPE embedding.
4. If samples have not been fixed for 36 hours, please leave them in 10% NBF or 4% PFA and inform HistoWiz customer service so we can transfer them to 70% ethanol after they arrive at HistoWiz.
5. This procedure ensures the best staining results for immediate projects and potential advanced staining in the future. See detailed fixation instructions

OCT Embedding can be compatible for H&E or SS staining, we recommend treating your samples with cryoprotectant solution following protocol below:

1. Fix your tissue in 10% Neutral Buffered Formalin or 4% PFA for 24–36 hours at room temperature.
2. After fixation, transfer your samples to a 15% sucrose in PBS until tissue sinks. Keep tissue at 4C during this step. ,
3. Transfer the sample to 30% sucrose and ship it overnight.

1. Cell Detachment: Rinse the culture with D-PBS, add pre-warmed trypsin, and incubate at 37°C until the cells detach. Collect the cells and transfer them to a centrifuge tube.
2. Centrifugation: Spin cells at 100 × g for 5 minutes, discard supernatant, resuspend cells in 1xPBS, and repeat centrifugation as above.
3. Fixation: Remove 1xPBS and fix the cells by adding enough (2-5ml, for 1-3 million cells). Adjust PFA volume for larger cell counts. Fix at room temperature for 15-20 minutes Final Centrifugation: Spin, discard fixative, resuspended in 1xPBS, and transfer cells to a 1.5ml tube (2-5 million cells) or 15ml tube for larger samples.
4. Storage and Shipping: Store in 1X PBS at 2-8°C for up to 1 week. Ship overnight with an ice pack.

1. Harvesting: Collect organoids in a 15 mL tube, centrifuge for 5 minutes at 200 × g, and remove media.
2. Rinsing: Rinse with 1X PBS, centrifuge for 5 minutes at 200 × g, and remove PBS.
3. Fixation: Fix in 4% PFA for 1 hour at room temperature or overnight at 4°C.
4. Post-Fixation: Centrifuge, remove fixative, wash with 1X PBS, and centrifuge again. Transfer to a 1.5 mL tube with 1 mL of PBS or 15ml tube for larger samples.
5. Storage and Shipping: Store in 1X PBS at 2-8°C for up to 1 week. Ship overnight with an ice pack.

1. Washing: Remove medium, wash the transwell twice with 1X PBS for 3-5 minutes at room temperature, and dab excess PBS.
2. Fixation: Add 4% PFA to both chambers, fully covering the cells. Incubate for 15-30 minutes at room temperature or overnight at 4°C.
3. Post-Fixation: Remove fixative, dab excess, and wash twice with 1X PBS for 3-5 minutes each.
4. Storage: Add 1X PBS to both chambers, wrap in parafilm, and store at 2-8°C for up to 1 week.
5. Shipping: Ship overnight in 1X PBS with an ice pack. Place each transwell in a tube filled ¾ with PBS.

1. Vial Labeling: When your samples are ready to be sent to HistoWiz, begin preparing your vials or containers for shipment. Ensure that your containers/cassettes/vials are appropriately labeled with the sample's names using commercial resistance ink or pencil.
2. Preparing Specimens: Place each organ/specimen in a separate vial or cassette. Avoid submitting multiple organs in the same container for best results. Ensure tissue to fixative volume ratio is 1:20.
3. Cassette Sizing: If sending tissue in cassettes, our standard tissue cassette for FFPE processing is 3 × 2.5 × 0.4 cm. We recommend using biopsy cassettes (1 x 1 mm2 square holes) for small samples and biopsies, like mouse glands or ovaries approximately 3mm in size. (figure 1)
4. Water-tight Receptacle: Place the cassettes in a water-tight jar and seal the lid with parafilm. Ensure that vials are also sealed.

1. Labeling: Ensure your FFPE block samples are labeled appropriately to the information provided in your HistoWiz order form. This will ensure the smooth processing of your order.
2. Packing: Place FFPE blocks in a ziplock bag, paper/plastic box, or cardboard sleeve. Close the bag and tape any open ends to secure the blocks (figure 1). Wrap the ziplock bag, paper/plastic box, or cardboard sleeve in bubble wrap, taping the ends to secure the bubble wrap in place (figure 1a).

1. Labeling: Ensure that your slides are labeled appropriately to the information provided in your HistoWiz order form. This will ensure the smooth processing of your order.
2. Packing: Place slides in a slide storage box. Do not send slides in other types of containers that may damage the slides in transit.
3. Securing Slides: Once packed, cover the slides with a strip of colored tape (Figure 1). The colored tape is put over the slides to prevent them from falling and keep them secure. Place one layer of folded paper towel on top of the slides (Figure 2) to ensure they do not shift around in shipment.
4. Sealing the Box: Once the box is closed, tape the outside. This ensures that the box won’t open in transit.

Please note: Sticky items like glue, tape, and bag edges will not hold in dry ice. Secure packing material with rubberbands to avoid damage during transit. Dry Ice: Include at least 10 lbs of dry ice for every 3 lbs of research material.

1. Packing Frozen Blocks or Frozen Tissue: Place Frozen blocks or cryovials in a ziplock bag or a dry ice-safe storage box.

• Securing packaging: Close any open ends and tie a rubber band around the bag or box to secure the blocks or cryovials. Wrap the ziplock bag, paper box, or cardboard sleeve in bubble wrap and tie a rubber band around the bubble wrap.

2. Packing Frozen Unstained Slides: Place slides in a slide storage box. Do not send slides in other containers that may damage them in transit.

• Protective packaging: Once packed, place one layer of thin bubble wrap on top of the slides (Figure 2) to prevent them from shifting during shipment.
• Securing Packaging: Once the box is closed, tie a rubber band around the outside. This ensures that the box won’t open in transit.

1. Aliquoting: We ask for a 40-50ul aliquot of the antibody along with the datasheet and positive/negative control samples.
2. Temperature control: Use ice packs or dry ice as needed to ship antibodies overnight.

The packaging can generally consist of three components:

1. Primary Specimen Containers: Ensure that samples are covered in fixative with little air in the container. Ensure your primary sample containers (like the sealed plastic containers, a ziplock bag of FFPE blocks, vials, or a box of slides) are closed and secured for transit.
2. Secondary Watertight Receptacle: If needed, wrap containers in parafilm and place specimen containers in a ziplock bag or another watertight container to prevent liquid spillage during transit.
3. Protective Layer: Wrap containers in bubble wrap to prevent movement or damage. If needed, the absorbent material can also be placed between the primary sample container and the outer packaging.
4. Outer Box: Place wrapped specimens in rigid cardboard or styrofoam outer packaging. At least one surface should be at least 100 mm x 100 mm. Add more bubble wrap or absorbent material as needed inside the box to cushion samples and prevent movement.
5. Ice Packs: Include ice packs in the shipment to prevent any melting of samples in transit, especially if you are sending cell cultures, spheroids, or antibodies

The packaging can generally consist of four components:

1. Primary Specimen Containers: Ensure your primary sample containers (like sealed plastic containers, a ziplock bag of FFPE blocks, vials, or a box of slides) are closed and secured for transit
2. Secondary Watertight Receptacle: If needed, wrap containers in parafilm and place specimen containers in a ziplock bag or another watertight container to prevent liquid spillage during transit.
3. Protective Layer: Wrap receptacles in bubble wrap to prevent movement or damage.
4. Dry Ice: Dry ice should be added to the bottom and the top of the box to surround the specimens and keep them frozen in transit. Please include at least 10 lbs of dry ice for every 3 lbs of research material.
5. Outer Box: Place wrapped specimens in rigid cardboard or styrofoam outer packaging. At least one surface should be at least 100 mm x 100 mm. Add more bubble wrap or absorbent material as needed inside the box to cushion samples and prevent movement.
6. Priority Overnight Shipping: Ship samples using priority overnight shipping methods. This will ensure that your samples are received and transferred to our -80 degrees celsius fridge as quickly as possible.

Generating a shipping label with HistoWiz can be done with one click directly on the order form.

• Enter a new shipping address on the order form and choose it from the dropdown list
• Select “HistoWiz generated FedEx label”. HistoWiz provides free inbound shipping for all orders over $500.
• Choose either overnight shipping.
• Click “Create Label” and then “Download Label” to print the PDF shipping label. You can also email this shipping label to a collaborator if they will be shipping your samples.

You can use your own shipping label by selecting “Ship it Myself”. If you're using your own label, please make sure to indicate the 5-digit HistoWiz order # on the package and provide us the Fedex tracking # in the Special Instructions field. You can also drop it off at our lab or one of our NYC dropbox locations.

1. Travel time: The transportation of your samples from your origin country to HistoWiz in New York City typically takes 2 to 4 days.
2. Checkpoints during Transit: Several organizations (e.g., international customs) will review the shipment for export from your country and import into the USA. Following proper HistoWiz packaging guidelines and providing complete documentation can promote faster processing and delivery.
3. Additional Information: Please note that during the shipment, you may need to provide documentation to get approval for export out of your home country and import into the US. This is a common practice may delay your shipment by a few days.