14 In Situ Hybridization for RNA: Nonradioactive Probe: RNA Probe
203
an anti-sense orientation are specific even though the labeled
cRNA with a sense orientation showed reactivity.
• Prolonged incubation time for alkaline phosphatase reaction
tends to yield non-specific reaction products. These should
not be taken as specific. Always be careful with the results
of negative control. In our experience the possibility of
non-specific reactivity is higher by overnight incubation of
alkaline phosphatase reaction.
• Proper fixation is preferable to avoid non-specific reactivity,
since the reaction time of alkaline phosphatase will be shortened considerably with a clear reactivity by adopting proper
fixation of, for example, 4% paraformaldehyde + 0.5% glutaraldehyde.
• Therefore, caution is required when one uses formalin-fixed,
paraffin-embedded conventional blocks. The author recommends using properly fixed blocks.
References
Motohashi H, Igarashi K, Onodera K, Takahashi S, Ohtani H, Nakafuku M,
Nishizawa M, Engel JD, Yamamoto M. Mesodermal- vs. Neuronal-specific expression of MafK is elicited by different promoters. Genes Cells
1996;1:223-238.
Ohtani H, Motohashi H, Sato H, Seiki M, Nagura H. Dual overexpression
pattern of membrane-type metalloproteinase-l in cancer and stromal
cells in human gastrointestinal carcinoma revealed by in situ hybridization and immunoelectron microscopy. Int J Cancer 1996;68:565-570.
Ohtani H, Nagai T, Nagura H. Similarities of in situ mRNA expression between gelatinase A (MMP-2) and type I procollagen in human gastrointestinal carcinoma: comparison with granulation tissue reaction. Jpn J
Cancer Res 1995;86:833-839.
Sasano Y, Furusawa M, Ohtani H, Mizoguchi T, Takahashi, T, Kagayama M.
Chondrocytes synthesize type I collagen and accumulate the protein in
the matrix during development of rat tibial articular cartilage. Anat Embryol 1996;194:247-252.
General advice for this
method
203
an anti-sense orientation are specific even though the labeled
cRNA with a sense orientation showed reactivity.
• Prolonged incubation time for alkaline phosphatase reaction
tends to yield non-specific reaction products. These should
not be taken as specific. Always be careful with the results
of negative control. In our experience the possibility of
non-specific reactivity is higher by overnight incubation of
alkaline phosphatase reaction.
• Proper fixation is preferable to avoid non-specific reactivity,
since the reaction time of alkaline phosphatase will be shortened considerably with a clear reactivity by adopting proper
fixation of, for example, 4% paraformaldehyde + 0.5% glutaraldehyde.
• Therefore, caution is required when one uses formalin-fixed,
paraffin-embedded conventional blocks. The author recommends using properly fixed blocks.
References
Motohashi H, Igarashi K, Onodera K, Takahashi S, Ohtani H, Nakafuku M,
Nishizawa M, Engel JD, Yamamoto M. Mesodermal- vs. Neuronal-specific expression of MafK is elicited by different promoters. Genes Cells
1996;1:223-238.
Ohtani H, Motohashi H, Sato H, Seiki M, Nagura H. Dual overexpression
pattern of membrane-type metalloproteinase-l in cancer and stromal
cells in human gastrointestinal carcinoma revealed by in situ hybridization and immunoelectron microscopy. Int J Cancer 1996;68:565-570.
Ohtani H, Nagai T, Nagura H. Similarities of in situ mRNA expression between gelatinase A (MMP-2) and type I procollagen in human gastrointestinal carcinoma: comparison with granulation tissue reaction. Jpn J
Cancer Res 1995;86:833-839.
Sasano Y, Furusawa M, Ohtani H, Mizoguchi T, Takahashi, T, Kagayama M.
Chondrocytes synthesize type I collagen and accumulate the protein in
the matrix during development of rat tibial articular cartilage. Anat Embryol 1996;194:247-252.
General advice for this
method