A review of fluorochromes and the specialized microscope used in immunofluorescence techniques will begin this presentation. A typical human skin and kidney clinical specimen will be followed from receipt in the laboratory, through freezing, cryomicrotomy and staining. Direct and indirect IF procedures will be discussed in detail. Technical comments of the many steps required for this technology will be given to allow the technologist a better understanding of the detail of attention required for this technology.
Hark! The Herald Angels Sing - Vienna Boys Choir* - The Little Drummer Boy of finished slides will conclude this presentation. Today we will have several learning objectives.
We will review the proper handling of specimens, discuss the importance of attention to detail. This is very important in producing a quality product, all be it immunofluorescence IFor any laboratory technique.
We will look at reagents and supplies required for immunofluorescent staining, and have images to look at for antibody staining pattern recognition. What we're going to do is follow a specimen, follow our specimens from receiving to the final product. We will perform direct and indirect manual IF techniques on frozen tissue. There will be a brief discussion of formalin fixed, paraffin-embedded immunofluorescent technique.
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It one of the most simple, simplest of all immunohistochemical techniques to perform. It is filtered light that excites a fluorochrome, a fluorescing compound, that is attached to an antibody. There are many Cy labels, Cy2, Cy3 are quite popular, and many Alexa Fluor fluorescent labels ライカ - HOMM∃ - Cold Finger well.
Some advantages and disadvantages of immunofluorescence. Advantages, It's a very rapid process. It has strong clinical implications. A clinician can get results back on a biopsy much faster than submitting formalin-fixed material and waiting for the processing, cutting, staining, and so forth.
It's an easily performed technique, and there are many other clinical and research applications, especially using multicolor staining. We will just be discussing single staining in this presentation. It does require an investment and a specialty microscope. It does require a designated viewing area, ライカ - HOMM∃ - Cold Finger in a darkened area, a darkened ライカ - HOMM∃ - Cold Finger . It requires a microtome, a cryomicrotome, and you do need a technologist that has the ability to perform antibody dilution.
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- Various - Furry Life! fluorescent-labeled antibodies. And the clinical application, it does require a control tissue bank, and an ultra-low freezer for storage of antibodies and tissue blocks.
And it does require your institution to be committed to this technology. Here we have a fluorescence microscope. And then a laptop or a PC to record and view the images as well. Use log showing bulb hours; some microscopes actually will have a clock that will record how many hours are on a bulb.
After a period of time the bulb loses intensity and needs to be changed. You should always wear gloves and safety glasses when changing the bulbs. The gloves prevent oil from your finger getting on ライカ - HOMM∃ - Cold Finger bulb and etching into the glass of the bulb. Keep a scheduled maintenance report file for CAP, or your accreditation inspections. I found that a Congo red histology special stain is a good slide to keep around the microscope because it fluoresces so well, and it can be used for microscope alignment.
Photography in the old days was used, was performed with 35 mm film that then had to be developed; whereas, today digital imaging is so much easier. If the phone should ring, or you get interrupted, make you leave the objective on the tissue section with the light source on, it will burn that out. Do put multiple sections on the slide so you can find that optimal area for photography, for capturing the image.
A couple of notes on the history of immunofluorescence, the Fluorescence Foundation is a good source for reviewing fluorochromes and the history. Coons labeled the first antibodies in with fluorescein, thus giving birth to the field of immunofluorescence. And not much has really changed today since his early development of this technology. Most of our tissue specimens we want to receive in the fresh state.
In ライカ - HOMM∃ - Cold Finger clinical arena, the most commonly submitted specimens are skin, kidney, and then other biopsies you may receive are heart, lung, liver, and conjunctiva biopsies. Fresh specimens are immersed in a volume 20 X greater than the specimen size, and specimens can typically be held for up to one week at room temperature. There's no need to refrigerate these specimens.
Here are two examples of dividing a renal biopsy. So you always want to handle the sections for immunofluorescence first to prevent contamination from your formalin or your glutaraldehyde for electron microscope EM samples. Always clean your forceps. The most common embedding media is OCT optimal cutting temperature compoundand you should have a cryostat chuck with a bed of OCT already frozen sitting in the bottom of the cryostat on the freezing bar.
Place your sample on there, put more OCT on top of it, and use the heat extraction bar in the cryostat to rapidly freeze that block. Another freezing technique is using liquid nitrogen-cooled methyl butane. It's the same technique you use for ライカ - HOMM∃ - Cold Finger muscle biopsies. Dry ice acetone slurry is another low-temperature freezing alternative, and there are commercial freezing baths and proprietary solutions as well.
I have a gelatin listed here as an embedding media, and I used that at Duke for 34 years, and actually it was used before my time in the immunofluorescence lab for many years prior to that. I will go into detail more on the gelatin embedding in just a moment.
So you want to make sure your renal biopsy specimen pieces are embedded close to each other. That allows you to trim a block, giving a smaller cut surface area. That allows you to group your sections on the slide in a much tighter group, thus making it easier for the pathologist to locate the slide of the sections, makes it easier for you when handling the sections, and also the amount of antibody you need to put on the slide to cover the sections.
As with all histologic technique, the skin is embedded on edge. A few comments about the embedding gelatin, you do not want to use house, kitchen-type gelatin, you want to use a Bloom strength, which I actually had that on the next screen, but a rapid freezing technique, such as the isopentane and liquid nitrogen must be used.
You do not want to use a slow freezing technique, like in a cryostat on the cold bar Keenan & Anderson* Feat. Tiff Lacey - Runaway for OCT. The gelatin will take on a totally different consistency and won't cut, so you must use a rapid-freezing technique.
Small embedding molds must be used. Adjust your cryostat temperature to minus 15 to 16, and once you get the knack of cutting gelatin you can take serial sections. They're so easy, they just Room With A View - Arcade - A/2 right off.
So, 7. Dissolve this mL of water, mL of water on Communist Limit - Seven Minutes Of Nausea - Our Culture Is Boring hot plate with a stir bar, add your 7. I would keep a ライカ - HOMM∃ - Cold Finger plastic bottle with gelatin sitting on a hot plate, not boiling hot, but just warm enough to keep it liquified, or you can keep the gelatin in a water bath to keep it liquified.
So, we've frozen our tissue, received our tissue, and have frozen it, and now we're ready to cut it in the cryostat. You want to prepare your slides, pre-label your slides with accession number, antibody, patient name, whatever your institution or lab requires. Use a permanent ink marker, because pencil will give graphite debris and it will smudge during handling. Like I said, trim your block down to a smaller size. Cut at 3 to 4 microns, and maintain order throughout the entire process, during your cutting, staining, cover-slipping, placement in the folder.
It helps with trouble-shooting, Rättviks Spelmanslag - Svensk Folkmusik maintaining order in the whole process. If you snap freeze in a low-temperature freezing system, allow your blocks to acclimate to the cryostat temperature. They can be too hard and need to acclimate. So use a slow, even cutting stroke, and I use room-temperature slides to pick up the sections.
You want to quickly pick up the sections and keep the frost wiped up on the blade holder. If the frost builds up on the blade holder you can have your sections stick to it, and then when you lift them up it causes a stingy, distorted artifact on your tissue sections. Again, place multiple sections on your slide and group them close together.
This illustrates a cutting sequence. I have slides labeled, sitting on top of ライカ - HOMM∃ - Cold Finger cryostat. That way you have multiple levels. This does show a pre-cut positive control on each of these slides. More on controls later. Here's an example of a polychrome methylene blue on a frozen section of kidney. Our glomerulus, the mesangium is this matrix inside the glomerulus. Like I said, this is a very rapid stain for frozen sections.
It's available commercially, but it's also easy to make for itself. It improves with age, like a fine wine.
You do want to filter before use to remove any particulates. It is a little stinky when you make it up, but it's worth making yourself. You get a much better product. The procedure is you cut your section, pick it up on a slide, dip the slide in the stain for five seconds, rinse the excess stain from the slide with water, and then you can either wipe the back of Lonely Child - Styx - Equinox slide and look at it under the scope as is, or you can put a cover glass on the slide, mounting with water.
And the sections are not permanent. Here is my frozen section fixative formula. It contain methanol, formaldehyde, and acetic acid. For optimal results, cut your frozen section and immediately pick it up, and immediately place it into the fixative for 60 to 75 seconds.
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