Possible reason:The glass slide is not cleaned before the sample is made

Recommended solution:Wash the glass slide with absolute ethanol

Possible reason:Washing is insufficient after hybridization

Recommended solution:Ensure that the PH value and temperature of the washing liquid are correct, remove the cover glass, and wash repeatedly

Possible reason:The filter group is used improperly

Recommended solution:Replace the appropriate filter group to observe to reduce the background light

Possible reason:The washing temperature is too low

Recommended solution:When the glass slide is washed, ensure that the temperature of solution reaches the temperature required in the LBP’s manual 

Possible reason:The sample is not well fixed

Recommended solution:Try to replace with other paraffin blocks for treatment, or recommend pathologists to communicate with clinicians in time for fixation time and effect

Possible reason:The washing liquid is used for a long time or stored incorrectly

Recommended solution:Ensure the use frequency of washing liquid, replace the washing liquid in time, and store the washing liquid according to the requirements of LBP’s manual

Possible reason:Samples are denatured insufficiently

Recommended solution:Ensure that the denaturation temperature of the hybridizer reaches the temperature required by the LBP’s manual, and the hospital needs to calibrate the equipment regularly

Possible reason:The probe is not sufficiently mixed before use

Recommended solution:Blow and beat the probe, mix well, and centrifuge shortly

Possible reason:The probe is not added

Recommended solution:Fully thaw the probe, to ensure that the pipette absorbs the reagent in the probe 

Possible reason:The probe is expired

Recommended solution:Use the probe within the validity period

Possible reason:The amount of the probe is insufficient

Recommended solution:Ensure that the pipette sucks accurately, and increase the amount of the probe to 10 ul. Please do not dilute the probe

Possible reason:Bubbles are formed under the cover glass during hybridization

Recommended solution:The cover glass should cover the surface of the probe and tissues when placed , and should be squeezed gently to squeeze out bubbles

Possible reason:Hybridization conditions are unsuitable

Recommended solution:Set according to the requirements of LBP’s manual

Possible reason:The temperature of the hybridizer about digesting,washing and overnight are incorrect

Recommended solution:Ensure that the equipment such as the water bath meets the temperature required in LBP’s manual

Possible reason:Washing liquid or washing conditions are incorrect

Recommended solution:Set according to the requirements of LBP’s manual

Possible reason:Sample glass slides or probes are stored incorrectly

Recommended solution:Set according to the requirements of LBP’s manual

Possible reason:Counterstain is used incorrectly or expired

Recommended solution:Use DAPI within the validity period, set and use DAPI according to the requirements of LBP’s manual

Possible reason:The filter group selected during observation is inappropriate

Recommended solution:Recommended to use the parameter settings of the reagent filter required in LBP’s manual

Possible reason:The sample is not well fixed

Recommended solution:Try to replace with other paraffin blocks for treatment, or recommend pathologists to communicate with clinicians in time for fixation time and effect

Possible reason:The front and back of the glass slide are error

Recommended solution:Retreat, and pay attention to the front and back when the glass slide is taken out

Possible reason:Glass slides are dried insufficiently

Recommended solution:Before the probe drops the reagent onto the glass slide, guarantee that the ethanol solution on the glass slide has been fully volatilized

Possible reason:The film-sealing gum or cover glass are not removed

Recommended solution:Remove all film-sealing gum in time and uncover the cover glass with moderate strength

Possible reason:Proper film-sealing gum is not used

Recommended solution:Use special Fish Fixogum film-sealing gum. Do not use sealing film, transparent adhesive top, and preservative film

Possible reason:The service life of microscope mercury lamp is expired

Recommended solution:Please contact the microscope manufacturer to replace with a new mercury lamp

Possible reason:The microscope structure and objective lens are not appropriate for observing FISH samples, or the filter is oxidized

Recommended solution:Please contact the microscope manufacturer

Possible reason:Glass slides are not pretreated before fixation, resulting that samples are the adhered with glass slides

Solution:Use glass slides with adhesives

Possible reason:Enzymatic activity of endogenous peroxides is not completely blocked

Solution:Block with 3% H2O2

Possible reason:Non-specific dyeing

Solution:Add 10% normal goat serum before adding antibody

Possible reason:The antigen is diffused due to autolysis of tissues

Solution:Use fresh tissues or fixed tissues in time as far as possible

Possible reason:Incomplete deparaffinating

Solution:Replace xylene and alcohol

Possible reason:Sections are flushed insufficiently

Solution:Flush completely

Possible reason:Enzyme reaction accelerates at too high room temperature

Solution:Ensure that the room temperature is 15-25℃ to shorten the developing time

Possible reason:Sections are dry during dyeing

Solution:Ensure that sections are moist

Possible reason:The antigen is denatured or blocked in the fixing or embedding process

Solution:It is recommended to use 10% neutral buffered formalin fixative;Ensure correct fixation time and baking temperature

Possible reason:The antigen is decomposed due to autolysis of tissues

Solution:Fix the samples in time