The accuracy of these graduations cannot be guaranteed. We suggest that they not be relied upon for measurement, but rather to be used for reference only.

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Yes. Put the slide in a Coplin jar or beaker filled with warm (37^oC) PBS buffer and let it sit, no agitation is required. The hardened ProLong mountant will swell and may slide off or be easily dislodged. If cells are adherent to the coverslip, make certain the coverslip side containing the cell or tissue sample does not land face down in the container or become scratched upon handling. Remove the coverslip, wash a couple of times, and proceed with re-staining and re-mounting in new ProLong mountant.

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The "Water Trap" should be flushed weekly.

1) Fill a clean 20mL, all-plastic Luer lock syringe (Cat. No. 4324463) with clean distilled or deionized water. Expel any bubbles. NOTE: Do not use a syringe smaller than 20mL or excessive pressure may be generated.

2) Attach the syringe to the forward-facing Luer fitting. Hold the fitting with one hand while threading the syringe onto the fitting with the other hand.

3) Open the forward-facing Luer fitting by grasping the body of the fitting and turning it and the syringe approximately one-half turn counterclockwise.

4) Open the exit fitting at the left side of the pump block by turning it approximately one-half turn counterclockwise.

5) Hold an empty beaker under the left side fitting while pressing gently but steadily on the syringe plunger. IMPORTANT: Take approximately 30 seconds to flush 5mL of distilled or deionized water through the trap. Excessive pressure can damage the "Water Trap" seals. Make sure that the exit/waste seals are open, in order to avoid pressure buildup. Continue to gently flush the full 20ml volume through the trap.

6) Close the forward-facing Luer fitting first (hand-tight only).

7) Close the left side exit fitting second (hand-tight only).

8) Remove the syringe from the forward-facing Luer fitting by holding the fitting with one hand while removing the syringe counterclockwise with the other hand.

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The Water Trap is located above the piston on the pump. There are 2 valves for the trap, one that points out towards you, the other points to the left. To clean the trap:

1) Loosen each of the valves by turning them half a turn counterclockwise.

2) Fill a 5 ml syringe with 4 ml of distilled water.

3) Attach the syringe to the valve extending towards you.

4) Holding a beaker under the valve pointing to the left, push through 3 ml of water. Do not completely empty the syringe to avoid introducing an excessive amount of bubbles into the Water Trap.

5) Tighten the valve pointing to the left and carefully remove the syringe. After removing the syringe, tighten the valve it was attached to. Note: The white end of the valve and grey body should not be separated. If you notice the white piece moving as you try to remove the syringe, re-tighten the syringe and secure the white piece before unscrewing the syringe.

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- Wearing gloves, apply sample (0.01-0.1 mL) using a pipette.
- OPTIONAL: Soak the Slide-A-Lyzer MINI Dialysis Unit to remove trace contaminants.
- Cap the Slide-A-Lyzer MINI Dialysis Unit (to minimize loss) and snap it into the float (sold separately).
- Insert the float into the beaker containing the dialysate.
- Recover sample.

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Yes, formamide is supplied as a liquid even though it is sold by weight.

It is appropriate to use out of the bottle for most applications (hybridization buffer, loading buffers for sequencing gels, and formaldehyde-containing RNA gels) if less than one year old and stored properly (-20 degrees C). (If formamide smells like ammonia, it is probably breaking down and should not be used.) However, we have seen that in a protocol we have for running RNA gels without formaldehyde in the gel, freshly deionized formamide is essential for preparation of the loading buffer. That protocol as well as the protocol for deionizing the formamide follows:

These reagents are used in electrophoresis of RNA in agarose gels in 1X MOPS EDTA buffer. (Note: no formaldehyde is used in the gel.) The sample is prepared in the sample buffer (1.5 to 3 µL sample mixed with sample buffer). Heat the RNA in sample buffer for 10 min at 65 degrees C. Place on ice immediately then load gel.

10X MOPS EDTA (10X ME)

(1) Measure out 700 mL DEPC-treated water in a DEPC-treated 1-liter graduated cylinder and place in a DEPC-treated 1-liter beaker.
(2) Place beaker on stir plate and begin stirring with a DEPC-treated stir bar.
(3) Add 104.65 g MOPS to beaker. Weigh out MOPS using a sterile-flamed spatula from an unopened bottle MOPS or one set aside for RNA only. Dissolve by stirring.
(4) Add 40 mL 0.25 M EDTA to solution. Note: the EDTA solution should be made with DEPC-treated processed water.
(5) Adjust pH to 7.0 by adding 10 N NaOH (~20 mL)
(6) Bring up to 1 liter volume with DEPC-treated water.
(7) Filter solution using a 0.2 µm Nalgene filter unit.
(8) Store at 4 degrees C for six months in a DEPC-treated glass bottle. (This prevents solution from yellowing.) NOTE: The presence of yellow color does not affect the performance of the buffer.

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One step of central importance in CISH is the incubation temperature during the heat treatment step. This represents the first antigen retrieval step in the CISH protocol. It is critical have the specimen be heated 98°C for 15 minutes in the pretreatment buffer included in the Spot-Light Tissue Pretreatment Kit (Cat. No. 008401) that we recommend. This can be accomplished in one of the following ways.

1) Microwave Oven: Place the slides in a plastic Coplin jar containing the pretreatment buffer and cap it loosely. Place a temperature probe in a separate jar containing water or buffer, but without a cap. Set the temperature to 93°C. Set the timer for 15 minutes when the temperature reaches 93°C. Note that the temperature in the jar with the cap should be ~98°C. Slides can be transferred to deionized water immediately afterwards. 

2) Pressure cooker: Once the water is boiling inside the cooker, set the timer for 10 minutes and apply the steam nozzle weight. Open the pressure cooker when the internal pressure has dropped to 0, at which point the temperature inside will be around 95°C. 

3) Hot plate: Heat the pretreatment buffer in a beaker. After the buffer temperature reaches above 98°C, put the slides in and heat them for 15 minutes. To prevent the buffer from evaporating, cover the beaker loosely with a glass cover or aluminum foil. 

4) Steamer: Make sure the pretreatment buffer temperature reaches 98-100°C before putting the slides in the container. Then, steam them for 20 minutes. 

It is important to note that the incubation time starts after the buffer is pre-heated to the required temperatures, not while it is heating up. 

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There should be continuous drip from the column and labeled proteins should elute within approximately 30 minutes or less. If the flow is slower or halted, this is most often caused by the frit becoming clogged with resin particulates. The frit is a small disc that sits on the bottom of the column to prevent the resin from flowing through but allows buffer and proteins to flow through. For the straw columns in our protein labeling kits, the frit is built into the column.
You should check the flow through BEFORE placing the labeled protein onto the column. When it slows down or becomes clogged, empty the resin into a clean, sterile beaker/container and rinse out the frit (shooting buffer in the opposite direction with a syringe) and repack the column.
If the labeled protein is stuck on the top of the column, collect only this fraction of resin (scoop it out) and repack to the top of a fresh column (with good flow through).
If you run out of resin or need more columns, the columns and resin are available (Antibody Conjugate Purification Kit, 0.5-1 mg conjugate, Cat. No. A33086)

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The procedure given below is a sample protocol for establishing cultures from the contents of one vial.

1. Prepare a beaker of water at 37 degrees C.
2. Remove a vial of cells from liquid nitrogen storage, taking care to protect hands and eyes.
3. Loosen the cap on the vial 1/4 turn for 10 seconds to release any liquid nitrogen that may be trapped in the threads, then re-tighten the cap.
4. Dip the lower half of the vial into the 37 degrees C water to thaw.
5. When the contents of the vial have thawed, wipe the outside of the vial with disinfecting solution and move to a Class II, type A laminar flow culture hood.
6. Open the vial and pipette the suspension up and down with a 1 mL pipette to disperse the cells.
7. Remove 20 µL from the vial and dilute the cell suspension in 20 µL of trypan blue solution (for example: Gibco Trypan Blue, Cat. No. 15250-061).
8. Use a hemacytometer to determine the number of viable cells per mL.
9. Dilute the contents of the vial (1 mL) to the concentration recommended by the product instructions (for example 1.25 X 10E4 viable cells/mL for Gibco neonatal human epidermal keratinocytes ).
10. Add 5 mL of cell suspension to each 25 cm² culture flask or 15 mL of cell suspension to each 75 cm² culture flask.
11. Following inoculation, swirl the medium in the flasks to distribute the cells. Many cell types attach to culture surfaces quickly, and if the medium is not distributed immediately following inoculation, the cells may grow in uneven patterns.
12. Incubate the cultures in a 37 degrees C, 5% CO²/95% air, humidified cell culture incubator. For best results, do not disturb the culture for at least 24 hours after the culture has been initiated.

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The Polymer Delivery Pump (PDP) and lower block, polymer channels, and tubing can and should be cleaned with all hardware components on the instrument. Run the Water Wash Wizard to clean the pump block, lower block, polymer channels, and polymer tubing. This should be performed monthly or as needed. Flush the Water Trap on a weekly basis by loosening the valves to the trap, attaching the syringe to the valve facing you and pushing water through while capturing the flow through in a beaker held under the other valve.

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