CFAR Immunology Core: Microscopy

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CFAR Immunology CoreMicroscopy

• Joy Folkvord
• joy.folkvord_at_uchsc.edu

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Whats AvailableAssistance with and resources
for

• Sample processing
• Staining
• Imaging (microscopy)
• Light microscopy
• Immunofluorscent
• Analysis

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Sample Processing

• Preparation
• Cells
• Cytosmears
• Chamber slides
• Frozen Cell pellets
• Tissue
• Frozen
• Cryostat available for cutting infectious
  specimens
• Formalin Fixed

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Staining

• Immunohistochemistry (IHC)
• Permanent
• Up to 3 colors
• Indirect staining
• Avidin/Biotin Protocols
• Hydrogen Peroxidase
• Alkaline Phosphatase
• Mouse on Mouse (M.O.M)

IHC for Ki 67 (purple) and CD8
Sample of M.O.M. - Mouse anti-human CD8 in Rag 2
mouse model
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Staining continued

• In situ hybridization
• HIV-1 producing cells
• Combine with IHC

Rag-II HIV positive mouse thymus a-sense
Rag-II HIV positive mouse thymus sense
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Staining continued

• Immunofluorescence (IF)
• Three colors (four?)
• FITC/AF488
• Texas Red/AF 594/PE
• Pacific Blue/DAPI
• CY5 (?)
• Colocalization
• Not permanent

Localization of neutrophils (CD15, green) in
lymphoid tissue in or outside of blood vessels
(Factor VIII, red) with nuclei stained with DAPI
(blue)
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ImagingLight Microscopy

• Leica DMR
• Digital color camera
• 2.5x, 10x, 40x and 63x (oil) lenses
• Bright Field
• Polarized Light
• Extensive Analysis Software

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Light Microscopy Image samples
40X - IHC stain of LN Red RANTES Blue-gray
CD20
40X - In situ (blue-black) and CD20 (brown)
double stain in Lymph Node
10X Hematoxylin and Eosin Stain of lymph node
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ImagingFluorescent Microscopy

• Leica DM5000B
• Digital BW camera
• 10x, 20x, 40x and 63x (oil) lenses
• Phase
• Fluorescent
• Some Analysis Software

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Fluorescent Images
63X a-defensin (red) and CD68 (green) in
cytosmear of LN cells
40X a-defensin (red) and CD15 (green) in LN
40X CD3 (red) and CD4 (green) in GALT
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Fluorescent Images (3 colors)
CD3
CD4
CD8
composite
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Image Analysis

• Sample Programs available
• Area
• Measure the area of the total tissue and/or
  structures of interest in a section of tissue
• Cells/area ( positive)
• Determine the proportion of a tissue crossection
  that is staining positive relative to the overall
  area
• Double positives

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Image analysis sample area
F
F
Quantification of the area staining positively
for a-defensin (red stain) in (F-green outline)
and out of (EF-yellow outline) lymphoid follicles
(blue-gray stain)
Follicle 3.8 Extrafollicular 12.8