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For my final project in the Goodwin College’s Histologic Science program I worked on skin specimens using Immunohistochemistry techniques and counterstained with Azure Blue. I was fortunate to complete my clinical rotation at Yale University School of Medicine Dermatology Department under the direction and guidance of Vincent Klump. I would like to thank Mr. Klump for suggesting this project and all of his help and leadership making my experience rewarding and valuable.
Azure Blue Counterstain vs. Hematoxylin to Distinguish Melanocytes from Melanin
By Emily Hast
Introduction
It is difficult to distinguish melanophages from heavily pigmented melanocytes when counterstained with hematoxylin. Azure B can be used as a counterstain instead to stain melanin blue-green. It can also be used alone as a special stain.
Skin stained with S100 and Azure Blue Counterstain Demonstrating Melanin brown and Melanocytes teal blue
Purpose
The use of Azure B makes the pathologist’s diagnosis much easier. Positive melanocytes will appear brown and melanin granules, cytoplasm, and negative melanocytes will be green-blue. Brown chromogen diaminobenzidene(DAB) is easily confused with melanin if Azure B is not used.
Skin stained with Melan-A and Azure Blue Counterstain
Demonstrating Melanin brown and Melanocytes teal blue
Method
Cut formalin fixed paraffin embedded tissue at 6 microns, adhere to aminosilane adhesive slides. Deparaffinize and run through graded alcohols to water. Perform antigen retrieval with HIER pH6 (DAKO catalog # 61699). Cool to room temperature, rinse and load into Labvision Autostainer. Ki-67 antigen(N1633RTU) is used to allow direct monitoring of the growth fraction of normal and neoplastic cells. It is also a reference antibody for the demonstration of the Ki-67 antigen. S100 (N1573RTU) is used in the identification of S100 positive neoplasms such as malignant melanomas, chondroblastoma, and schwannoma. Melan-A (N1622RTU) is a melanoma specific antigen transmembrane protein that is expressed in skin, retina and the majority of cultured melanocytes as well as melanomas and angiomyolipomas. If melanomas are ruled out it is then used to diagnose adenocortical carcinomas. Universal LSAB2 (KO675) is a peroxidase based visualization kit used with rabbit and mouse primary antibodies. The biotinylated link used is produced in goats. Liquid diaminobenzidine (DAB) when oxidized forms a stable brown end product at the site of the target antigen or nucleic acid. Upon completion on the autostainer slides should be rinsed quickly in tap water, Azure B counterstain should then be applied to slides for 15-45 minutes. A quick dehydration in ethanols follows along with clearing and cover slipping. Azure B can be used as a special stain to achieve the same results.
Skin stained with Melan-A(left) and S100(right) with Azure Blue Counterstain
Demonstrating Melanin brown and Melanocytes teal blue
Summary
When using routine hematoxylin as a counterstain in Immunohhistochemistry it is difficult to distinguish heavily pigmented melanocytes from melanophages. If Azure B is used a distinct difference in color can be seen. Brown chromogen DAB reaction product will not be confused with blue-green melanin stained with Azure B. Azure B is a superior counterstain, and special stain, for distinguishing melanophages from heavily pigmented melanocytes, making for an easier diagnosis.
Skin Epithelium demonstrating Melanin brown and Melanocytes teal blue stained with Melan-A and Azure Blue counterstain
Works Cited
C J Kligora, K P Fair, M S Clem, and J W Patterson “A comparison of melanin bleaching and azure blue counterstaining in the immunohistochemical diagnosis of malignant melanoma.” Pathol. 1999 December; 12(12): 1143–1147
Kamino, H, and S T Tam. “Immunoperoxidase technique modified by counterstain with azure B as a diagnostic aid in evaluating heavily pigmented melanocytic neoplasms.” Journal of Cutaneous Pathology 18.6 (1991) : 436-439.
Michelle Chapman
June 14, 2011
Laser Microdissection
SAFTY FIRST! The Microscope, as well as the Laser, is intended for indoor use only, all plugs should be plugged into a grounded socket, use away from any water sources, keep the microscope in a consistent temperature, and keep the laser encapsulated at all times.
The Laser Microdissection System is a combination of a microscope, laser,and PC that allow procurment of specific histologic structures in the form of single cells or groups of cells.
The Intended purpose of this system is for the preparation of the specimens for both biomedical research and the diagnosis of illnesses, by helping to improve the molecular examination methods done by isolating specific areas of tissue in a precise contamination free way, which can be readily assailable for further study and research.
Matierals that can be used are either human or animal tissue obtained onto a slide by frozen sectioning, smears, paraffin sections. The tissue can stained or unstained, and also the LCC extension module (living cell cutting) can be used for isolating living cells from cultures contamination free.
The specimen used was my own cheek cells swabbed and smeared onto a Membrane slide (PEN-Membrane 2.0 um) and then stained with Hematoxylin and Eosin. the PC and Microscope unit were turned on and the system loaded the prior configurations of the program. The slide and PCR caps (used for collection of dissected tissue), were loaded onto the microscope and the magnification (ranging from 10x to 30x) was focused using the x/y joystick. Once an appropriate visuale could be made out on the monitor, the laser was calibrated for the conversion of the coordinates of the mouse cursor into the coordinates of the laser beam. I then adjusted the speed and thickness of the laser in order to get a very precise cut around the tissue. Again, using the x/y joystick I selected different areas of tissue to cut out using the “Draw+Cut” mode, which allowed me to draw a specific area around the tissue before the laser cut it out and dropped it into the capsule. 4 to 5 areas were cut and were immeadiatly ready to be viewed inside the capsule.
In conclusion, I really enjoyed using the Laser Microdissection system, and I believe that in future research it will be highly beneficiary to research, and to diagnosis illnesses and causes of these illnesses.
