- Thaw slides for 30 s at room temperature by placing horizon-
tally on a clean surface (e.g., 5 mL pipette tips on plastic tray). - Place into 75% ethanol for 30 s (seeNote 6).
- For HistoGene staining: incubate slides in ddH 2 O for 30 s and
subsequently dry by briefly tilting them onto lint-free tissue
(skip this step for cresyl violet staining). - Place slides horizontally, cover with HistoGene or cresyl violet
staining solution (~200μL) for 20–60 s (seeNote 7), then
remove staining solution by tilting slides onto lint-free tissue. - For HistoGene staining: wash in ddH 2 O for 30 s (skip this step
for cresyl violet staining). - Dehydrate by moving slides through rising ethanol concentra-
tions (75, 95 and 99.7% ethanol, 30 s each) followed by tilting
the slides onto lint-free tissue. - Place slides into a dry, new slide box and initiate the LCM
procedure.
3.4 Rapid Antibody
Tissue Staining for
Laser Capture
Microdissection
Transport slides (in slide boxes) on dry ice. Use forceps to move
slides between staining containers, wear gloves and clean these reg-
ularly with RNaseZap. For examples of antibody stainingsseeFig. 2.Fig. 2Examples of tyrosine hydroxylase antibody staining and staining Nissl of human midbrain dopamine
neurons. Midbrain dopamine (mDA) neurons in the ventral tegmental area visualized using a (a–d) quick TH
antibody staining or (e–h) by using cresyl violet. (a–b, e–f) Low magnification images provide an important
anatomical reference that later aid in interpreting the corresponding sequencing data. (c, d) While the TH
antibody facilitates high specificity for laser dissection of dopaminergic cells, it lacks the ability to visualize
surrounding nuclei of small cells that could be a source of contamination. (g, h) In contrast, when the cresyl
violet is combined with the presence of neuromelanin, a feature of adult human midbrain dopamine neurons,
the staining also reveals the nuclei of small cells and could aid in enriching the target population. Scale bars in
e(applicable toa,e)¼ 2000 μm, inf(applicable tob, f)¼ 400 μm and inh(applicable toc, d, g, h)¼ 50 μm.
Human tissues were kindly received from the Netherlands Brain Bank (NBB) and the National Disease
Research Interchange (NDRI)102 Susanne Nichterwitz et al.