Table 2.

Relative labeling intensity for antibodies as a function of pretreatment protocol

Target (against)Quality of labeling
Steamer/citrateNo pretreatmentHClMicrowave (low)
BrdU42
PCNA40–10–24
Phospho-H3401–24
Pax642–32–34
p27Kip1400–13
S10043–42–34
Type III β-tubulin432–34
DAPI43–44
Hoechst 33342434
Syto 24444
  • 4, Equal or almost equal to label intensity with citrate/steamer pretreatment; 3, distinctly weaker than citrate steamer, but still quite satisfactory; 2, quite weak relative to citrate/steamer, but still satisfactory; 1, very weak, but some specific label detectable; 0, no specific label observed (at 10×); –, no specific label detected or marked reduction in labeled cell number, even when viewed at high power. Labeling intensity relative to steamer/citrate pretreatment was subjectively scored by comparison of images collected using a 10× Neofluar lens and an exposure time optimized for the steamer/citrate pretreatment slide of the same experiment. Within each experiment, serial sections of a single brain were labeled; thus, little variability is expected between slides with respect to tissue quality and fixation. For each antibody, labeling intensity was scored for each pretreatment in at least two separate experiments. In every experiment, steamer/citrate was superior to HCl and no pretreatment, but there was some variability between experiments in relative labeling intensity for some antibodies. When the scores varied, the range is indicated. In one experiment, the labeling intensity for Pax6 and S100 was slightly brighter for slides pretreated with the microwave (low) protocol compared with the steamer/citrate protocol. In all other cases, the steamer/citrate protocol produced labeling brighter than or equal to the microwave (low) protocol. PCNA, Proliferating cell nuclear antigen.