Collecting the following equipment and supplies is a crucial first step toward beginning your own lab. Refer to the protocols for more detailed information on how each of the following pieces of equipment is used in the immunostaining process. The recipes will detail which chemicals are necessary to prepare each reagent or solution. (click on each picture for a larger image)
1. Freezing-sliding Microtome (model HM440E from MICROM)
Used for sectioning skin biopsies after they have been fixed. The microtome is used in conjunction with the microtome stage and the microtome knife to provide a fully functioning biopsy sectioning solution.
2. Brass Microtome Stage
Used in conjunction with the freezing-sliding microtome to provide a well for dry ice and a surface for the sucrose stage to adhere. The sucrose stage will provide a buffer between the microtome stage and the knife of the microtome. Our stage was custom built at Johns Hopkins.
3. Microtome Knife
Used in conjunction with the freezing-sliding microtome to section skin biopsies. Knives are not included in the purchase of the microtome. All knives should be kept sharp on a regular basis.
4. Refrigerator(s) and Freezer(s) [-70 degree C, -20 degree C, and 4 degree C]
Used to store various reagents and specimens. All cooling equipment should be hooked up to emergency power outlets in case of a power outage. Freezers should be hooked up to an on-line temperature regulated system that notifies emergency telephone numbers in the event of a rise in internal temperature.
5. Binocular Dissecting Microscope (Zeiss STEMI SV8)
Used for transferring skin sections through the immunohistochemistry process. It is important to purchase a durable, reliable, comfortable model because this is the one piece of equipment that the greatest about of time is spent. It is also important that the scope have a top mounted, variable intensity light source and a double sided (black and white) base plate; but these are optional.
6. Shaker Table (Labline Model 600)
Used to agitate skin samples while in a particular solution for a long period of time. The shaker should have variable speeds and some method of securing 96 well plates (i.e. rubber mat, clamps, straps, etc.).
7. Hot plate / Stir plate
Used to mix chemicals and provide heat when making various solutions. The heat plate should be capable of generating at least 55 degrees Celsius.
8. pH Meter
Used for measuring the pH of various reagents. It is CRITICAL that the pH of each solution outlined in the recipes for creating the reagents and also specified in many of the protocols be EXACTLY as stated or the experiment may not work properly. The pH meter should also be capable of measuring the pH of Trisbuffer solutions.
Used to agitate various solutions. This is used when shaking and/or stirring a solution will not suffice. The vortexer is used most often when making the Oxalic Acid, Potassium permanganate, blocking, primary and secondary solutions described in the Recipes section and referenced in the immunohistochemical protocol.
10. Balance (Mettler, toploading)
Used to weigh dry chemicals such as Sodium m-Periodate. The balance must be readable to .01g in order to weigh certain chemicals within specified limits. The balance should also be capable of being calibrated externally via standard weights. Calibration should be performed and results recorded on a yearly basis.
11. Platinum Transfer Loops
Used in the creation of a transfer loop which is used to move sections from one well containing reagent to the next well with new reagent. It is important that the loop be platinum because it is a bendable metal that causes the least amount of secondary reactivity when introduced to the chemicals and reagents used in the immunohistochemical experiment.
1. Microcentrifuge (Eppendorf 5415)
used to spin tubes of antibody to ensure efficient usage of valuable drops of reagent.
2. Multi-head Microscope (Zeiss)
used to review the final skin biopsy slides after they have been successfully immunostained. The microscope is useful when multiple physicians/researchers collaborate on a given subject. Another useful feature is the ability to transmit the image to a monitor via a digital camera mounted on the master scope. The digital image can then be captured and used in a variety of applications.
3. Graphics Workstation
used to scan 35mm slides, produce photographic panels via photomanipulation in Adobe Photoshop, print images on various media, and assemble presentations using Microsoft PowerPoint. We have found our graphics workstation to be a valuable resource when assembling grants, presentations, and publication material.
used to store relevant information about each biopsy taken, demographics of the patient, billing procedure, experimental procedures, and diagnostic findings. A database is EXTREMELY useful when referencing previously collected data or tracking the current status of a particular biopsy.
|1. PGP 9.5 Polyclonal Antibody (Biogenesis)|
|2. Biotinylated Goat-Anti-Rabbit Secondary Antibodies (Vector)|
|3. Biotinylated Goat-Anti-Mouse Secondary Antibodies (Vector)|
|4. Vector SG Peroxidase Substrate Kit (Vector)|
|5. ABC Elite Kit (Vector)|
|6. Vectabond Subbing Solution (Vector)|
|7. Normal Goat Serum (Vector)|
|8. Anhydrous Glycerol (Sigma, Baker, or Fisher) 2136-03|
|9. Sodium Chloride (Sigma, Baker, or Fisher) S640-3|
|10. Trisma Base (Sigma, Baker, or Fisher) T-1503|
|11. Ethylene Glycol (Sigma, Baker, or Fisher) 9300-03|
|12. Concentrated HCl (Sigma, Baker, or Fisher) 9535-33|
|13. 50% w/v Sodium Hydroxide (Sigma, Baker, or Fisher) SS-254-500 41,541-3|
|14. 10N Sodium Hydroxide (Sigma, Baker, or Fisher)|
|15. L-Lysine (Sigma, Baker, or Fisher) L-5626|
|16. Potassium Permangenate (Sigma, Baker, or Fisher) P9810|
|17. Oxalic Acid (Sigma, Baker, or Fisher) 0-0376|
|18. Sodium Phosphate, Dibasic and Monobasic (Sigma, Baker, or Fisher) 3828-05/ 3818-05|
|19. 100% Ethanol (Sigma, Baker, or Fisher) Pharmco DSP-CT-18 203-|
|20. EDTA (Sigma, Baker, or Fisher)|
|21. Xylenes (Sigma, Baker, or Fisher)|
|22. Permount (Sigma, Baker, or Fisher)|
|23. Sodium m-Periodate (Sigma)|
|24. Paraformaldehyde [Prill Grade] (Electron Microscopy Sciences)|
|25. Powered Milk (Carnation)|
|1. 96-well Low Evaporation Culture Plates [3072-96] (Falcon)|
|2. Glass Slides and Coverslips (Sigma, Baker, or Fisher)|
|3. 17 x 100mm Snap Cap Tubes (Sigma, Baker, or Fisher)|
|4. Screw Cap Eppendorf Tubes (USA Scientific)|
|5. 50ml and 15ml Screw Cap Centrifuge Tubes (Fisher)|
|6. Parafilm (VWR)|
|7. Slide Trays and Slide Boxes (VWR)|
|8. Solvent-proof Histology Markers (VWR)|
|9. Fine Point Forceps (VWR)|
|10. Powder-free Gloves [UltraOne] (Microflex)|
|11. 3mm Skin Punch (Accuderm)|
|12. Disposable Forceps (Accuderm)|
|13. Disposable Scalpels (Accuderm)|
|14. Sterile Gauze Pads (Hospital Supply)|
|15. 2% Lidocaine with Epinephrine (Hospital Supply)|
|16. 1 inch Band Aids (Hospital Supply)|
|17. 1cc Syringes (Hospital Supply)|
|18. Paper Tape (Hospital Supply)|
The following protocols are ready to print and available for download:
|1. Immunohistochemical Staining of Human Skin Specimens.|
|2. Pete Hauer's Protocol for Subbing Slides.|
|3. Quantitation protocol.|
The following recipes are ready to print and available for download:
|1. 8% Paraformaldehyde Stock Solution|
|2. 0.4M Sorrensons Phosphate Buffer Stock (used at 0.1M on tissue)|
|3. Lysine Stock Solution|
|4. PLP Fixative|
|5. Cryoprotectant for Tissue Blocks|
|6. Antifreeze Solution for Tissue Blocks|
|7. Mowiol Fluorescent Mounting Medium|
The following forms are ready to print and available for download:
|1. Skin Biopsy Identification Form|
|2. Outside Billing Sheet|
|1. No biopsy should be in fixative longer than 24 hours. This is an important consideration when shipping specimens via Fed-Ex, UPS, or Airborne Express. Specimens should only be sent priority-one overnight.|
|2. No biopsy should be frozen until it has been sufficiently cryoprotected (a minimum of 12 hours). The cells that tissue is comprised of are filled with liquid and, if frozen before cryoprotection, will burst which will result in destroyed tissue.|
|3. The pH of all reagents that are made in the lab MUST be checked and MUST be exactly as outlined in the protocols and recipes because the pH directly affects the interaction of chemicals (particularly antibodies) during the staining process.|
|4. It is important to make up sufficient amounts of stock solutions before they are needed but it is also important to have fresh solutions as well. Avoid using "stale" solutions. As a general rule, we make paraformaldehyde stock and Lysine stock at the beginning of each week because we use the solutions on a daily basis.|
|5. Store unused sections in antifreeze solution for later use. We have stored and successfully retrieved and stained sections that were 10 years old using this method. Also make sure that if sections are stored in this manner over long periods of time precautions are taken to ensure the antifreeze levels stay constant.|
|6. Be sure not to mix antifreeze solution into a frozen biopsy during sectioning. It is easy to allow excess antifreeze solution to stay on the brush or on the knife when sectioning but remember that if antifreeze solution penetrates the frozen biopsy it will begin to thaw and the result will be irregular sectioning.|
|7. Try to prevent excessive vortexing of any solution containing anitobies because the antibodies are fragile by nature. Excessive vortexing may result in failed immunostaining due to the destruction of a majority of anibodies.|
|8. Remember that when preparing fluorescent labeled sections use Mowiol Mounting Medium. Also remember to cover the 96-well Plate with aluminum foil to prevent excess bleaching due to fluorescent light in the room.|