Konference: 2010 6. sympózium a workshop molekulární patologie a histo-cyto-chemie
Kategorie: Onkologická diagnostika
Téma: Postery
Číslo abstraktu: p013
Autoři: M. Vařejková; H. Hollerová; Z. Kudláčková; Prof.MUDr. Jaroslav Mokrý, Ph.D.
Standard immunohistochemical detection methods
make visualization of only two or three antigens in a histological
section. However, this approach does not allow us to exploit the
full potential of scarce archived paraffin-embedded sections. We
utilized a recently described sequential immunoperoxidase labelling
and erasing (SIMPLE) method (Glass et al. J Histochem Cytochem
2009,57:899-905) for simultaneous visualization of intermediate
filament nestin with other markers within a single tissue section
to investigate multiple antigen expression in the vasculature of
the uterine horns of pregnant rats.
Paraffin-embedded sections containing the structures of a interest were deparaffinised in xylen and rehydrated in series of graded alcohols to distilled water. Sections were exposed to microwaves to revitalize epitopes in tissue fixed with 4% paraformaldehyde and pretreated with normal donkey serum. Then the first antigen, intermediate filament protein nestin, was detected with Rat 401 antibody. After washing, donkey anti-mouse biotinylated antibody was applied followed by incubation with streptavidin conjugated with horse radish peroxidase. After washing, the signal was developed using aminoethylcarbasole and sections were mounted in buffered glycerol.
Following microphotography of nestin-immunoreactive structures with DP71 digital camera, the slides were decoverslipped, the red specific signal was washed away by ethanol and antigen-antibody complex was removed in an elution solution. The immunostaining process was then repeated in subsequent steps using the same protocol with distinct primary antibodies: anti-PCNA was used in the second round of immunostaining, anti-desmin in the third and anti-BrdU in thefourth round. Each time the immunopositive signals were examined with BX51 microscope and digitalized images were archived. We also modified the original SIMPLE method to test whether its principles is applicable to immunophosphatase labelling. In this modification, we utilized streptavidin conjugated with alkaline phosphatase and specific signal was developed with Fast Red TR/naphthol AS-MX tablets. To create multicolour composite images, digital snapshots of identical sites with distinct signals obtained in different rounds of the SIMPLE method were aligned as separate layers using Adobe Photoshop software and red colour of immunoreactive structures was replaced with the distinct pseudocolour.
Immunohistochemical staining provided evidence of nestin expression in endothelium, smooth muscle cells and interstitial myofibroblasts. In endothelial cells, nestin was intensely expressed in the arterial endothelial lining while it was mostly absent from lumina of adjacent veins. Endothelium of blood capillaries was also immunoreactive for nestin. Coexpression of nestin with proliferative markers PCNA an BrdU was observed in endothelial cells; its coexpression with desmin was typically observed in myocytes.
Our results provide evidence for different expression of nestin in arterial and venous endothelium which may be associated with remodelling of blood vasculature during pregnancy and the different functional demands on arteries and veins. We demonstrated the feasibility of SIMPLE method in investigation of multiple antigens expression in the remodelling of blood vessels. The SIMPLE method for immunoperoxidase proved to be less problematic than immunophosphatase staining due to formation of precipitate after antibody elution.
Paraffin-embedded sections containing the structures of a interest were deparaffinised in xylen and rehydrated in series of graded alcohols to distilled water. Sections were exposed to microwaves to revitalize epitopes in tissue fixed with 4% paraformaldehyde and pretreated with normal donkey serum. Then the first antigen, intermediate filament protein nestin, was detected with Rat 401 antibody. After washing, donkey anti-mouse biotinylated antibody was applied followed by incubation with streptavidin conjugated with horse radish peroxidase. After washing, the signal was developed using aminoethylcarbasole and sections were mounted in buffered glycerol.
Following microphotography of nestin-immunoreactive structures with DP71 digital camera, the slides were decoverslipped, the red specific signal was washed away by ethanol and antigen-antibody complex was removed in an elution solution. The immunostaining process was then repeated in subsequent steps using the same protocol with distinct primary antibodies: anti-PCNA was used in the second round of immunostaining, anti-desmin in the third and anti-BrdU in thefourth round. Each time the immunopositive signals were examined with BX51 microscope and digitalized images were archived. We also modified the original SIMPLE method to test whether its principles is applicable to immunophosphatase labelling. In this modification, we utilized streptavidin conjugated with alkaline phosphatase and specific signal was developed with Fast Red TR/naphthol AS-MX tablets. To create multicolour composite images, digital snapshots of identical sites with distinct signals obtained in different rounds of the SIMPLE method were aligned as separate layers using Adobe Photoshop software and red colour of immunoreactive structures was replaced with the distinct pseudocolour.
Immunohistochemical staining provided evidence of nestin expression in endothelium, smooth muscle cells and interstitial myofibroblasts. In endothelial cells, nestin was intensely expressed in the arterial endothelial lining while it was mostly absent from lumina of adjacent veins. Endothelium of blood capillaries was also immunoreactive for nestin. Coexpression of nestin with proliferative markers PCNA an BrdU was observed in endothelial cells; its coexpression with desmin was typically observed in myocytes.
Our results provide evidence for different expression of nestin in arterial and venous endothelium which may be associated with remodelling of blood vasculature during pregnancy and the different functional demands on arteries and veins. We demonstrated the feasibility of SIMPLE method in investigation of multiple antigens expression in the remodelling of blood vessels. The SIMPLE method for immunoperoxidase proved to be less problematic than immunophosphatase staining due to formation of precipitate after antibody elution.
Study was supported by MSM 0021620820.
Datum přednesení příspěvku: 23. 4. 2010
