Optimized fixation and storage conditions for FISH analysis of single-cell suspensions.
In our protocol to isolate and identify fetal cells in maternal peripheral blood, antibody (Ab)-stained cells are preserved with paraformaldehyde (PF) before batch flow cytometric sorting. However, PF fixation compromises the quality of subsequent interphase fluorescence in situ hybridization (FISH). We therefore examined the effect of PF concentrations and storage time in phosphate-buffered saline (PBS) on the quality of FISH signals. Cells were analyzed for changes in light scatter, morphology, and accessibility of target cell DNA. Fixation in 3% PF for 1 hr was ideal for both flow cytometry and subsequent FISH detection. However, beyond 10 days of storage, FISH quality deteriorated. (J Histochem Cytochem 46:971-973, 1998)
Murrell-Bussell
S
S
Departments of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.
Nguyen
D
D
Schober
W D
WD
Scott
J
J
Simpson
J L
JL
Elias
S
S
Bischoff
F Z
FZ
Lewis
D E
DE
D002021:Buffers; D005333:Fetus / Q000166:cytology; D005404:Fixatives; D005434:Flow Cytometry; D005557:Formaldehyde; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D011108:Polymers; D013048:Specimen Handling; D013997:Time Factors; D016707:Tissue Fixation
D002021:Buffers; D005404:Fixatives; D011108:Polymers; D005557:Formaldehyde; C003043:paraform