QBI HISTOLOGY AND MICROSCOPY GUIDE

Project Pathways: Fluorescence Mouse Tissue Sections
What do you want to see?
High resolution
Example: localising a synaptic
marker or looking at intracellular
proteins
Cell Numbers
Example: counting neurons within
the hippocampus
Gross Morphology
Example: looking for gross
changes in anatomy or cell
densities
Fix tissue using trans-cardial perfusion
Section on cryostat (2-20µm) or paraffin
block (4-10µm)
Fix tissue using trans-cardial perfusion
Section on vibratome or sliding
microtome (40 - 50µm)
Fix tissue using trans-cardial perfusion
Section on vibratome or sliding
microtome (40 - 50µm)
Recommended:
DAPI + GFP/Alexa488 + Alexa594
or Alexa647
Try to avoid using markers prone
to bleedthrough
Count all cells?
DAPI - BUT easier to use a
brightfield technique
All cells + specific cells?
DAPI + Alexa488, Alexa555,
Alexa647
Recommended:
DAPI + GFP/Alexa488 + Alexa594
or Alexa647
Try to avoid using markers prone
to bleedthrough
Imaging:
Preferably image in 3D on an
LSM confocal. - use Apotome as
a minimum.
Analysis:
Use Imaris to analyses and
visualise your data
Qualitative
1. Mount in prolong gold
2. Image using 20-63x objective
3. Density analysis on images
Quantitative
1. Mount in prolong gold
2. Seal slides with clear nailpolish
3. Stereological Analysis
Imaging:
Image on AxioImager Green or
Blue using MosaiX at 10-20x
or
Image on Vslide at 20x (if imaging
large quantities or for better
stitching results)
or
Image on LSM Confocal
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