"Red prefers dry but green prefers wet"in fluorescence intensity during FISH detection

PAN Yiqing; DING Lihong; ZHENG Jie

doi:10.3969/j.issn.1007-8096.2025.09.009

Abstract：Objective To compare the fluorescence intensities of red and green signals in fluorescence in-situ hybridization(FISH)under different humidity conditions,and to explore the humidity preference of fluorescence intensity in FISH detection.Methods Nine specimens each of breast cancer and lung cancer tissues were collected.Each specimen was serially sectioned into 7 slices.One slice was stained with HE to identify the tumor area,and the remaining 6 slices were divided into two groups(3 slices per group).Probes from two different manufacturers were added dropwise to each group,respectively.The 3 slices in each group were hybridized overnight under different humidity conditions,with the water addition to the wet strip in the in-situ hybridization instrument set at 5 ml,15 ml,and 25 ml.On the following day,the hybridization results were counted and interpreted under a fluorescence microscope,and the average fluorescence intensity of the post-hybridization images was calculated by using Image J software.Statistical analysis was performed on the fluorescence intensities of red and green signals from FISH probes under three humidity gradients to explore the influence of different humidity conditions on the red and green fluorescence intensities.Results There was no significant difference in the success rate of the hybridization detection between the two tissue types under different humidity gradients(P＞0.05),but there was a statistically significant difference in the fluorescence intensities of red and green signals under different humidity conditions(P＜0.05).Both red and green fluorescence signals were strong under high hybridization humidity and weak under low hybridization humidity.Conclusion Humidity has a great influence on the intensities of red and green fluorescence signals.During the FISH detection process,it is necessary to ensure a humid environment during hybridization.A humid hybridization condition is more conducive to obtaining stronger red and green fluorescence signals,facilitating counting.Additionally,when the red signal is weak,moderately increasing the hybridization humidity can help obtain clearer hybridization results.