This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.
The overall goal of this procedure is to localize low level expressed odorant receptor genes as well as other genes in the insect antennae using digoxigenin labeled or biotin labeled probes. To begin this procedure, select the new molting adult locusts that are active and have intact antennae, then cut the antennae into two to three millimeter pieces using sterile razors. Next, apply OCT compound on a freezing microtome holder and place the samples on the compound horizontally.
Transfer the holder into the freezing microtome at negative 24 degrees Celsius to equilibrate until the compound freezes. Afterward, take out the holder and cover the samples with a little compound. Transfer the holder into the freezing microtome at negative 24 degrees Celsius again for at least 10 minutes, then section the frozen samples into 12 micrometer thick slices.
Subsequently, thaw out the slices one by one on slides and air dry them for 10 minutes. After preparing the cryostat sections, place the slides in a plastic container and fix the tissues by incubating the slides in 4%PFA solution for 30 minutes at four degrees Celsius. After 30 minutes, wash the slides in one X PBS for one minute.
To eliminate the alkaline proteins, transfer the slides to 0.2 molar hydrogen chloride for 10 minutes, then transfer the slides to one X PBS with 1%Triton X-100 for two minutes to eliminate the surface protein of nucleic acid. Next, wash the slides twice for 30 seconds in one X PBS. Subsequently, rinse the slides in formamide solution for 10 minutes at four degrees Celsius.
Drain the slides and add 100 microliters of diluted antisense and sense probes, then place the cover slips on the tissue sections. Afterward, add formamide solution or one X PBS to the bottom of the box to keep the environment moist, but do not submerge the slides in the liquid. Following this, place the covered slides horizontally in a humid box and incubate at 55 degrees Celsius for 22 hours.
After hybridization, remove the cover slips carefully. Wash the sides twice by gently agitating them on a rocker for 30 minutes in 0.1 X SSC at 60 degrees Celsius, then rinse the slides in one X TBS for 30 seconds. Add one milliliter of 1%blocking reagent in TBS supplemented with 0.03%Tritin X-100 on each slide and incubate for 30 minutes.
Afterward, discard the blocking solution. For chromogenic detection, use blocking reagent in TBS to dilute 750 units per milliliter anti-digoxigenin-AP conjugated antibody to 1.5 units per milliliter AP solution. Subsequently, add 100 microliters of AP solution to each covered slide.
For TSA detection, use blocking reagent in TBS to dilute 750 units per milliliter of anti-digoxigenin-AP conjugated antibody and anti-biotin streptavidin HRP conjugated antibody to the AP HRP solution, then add 100 microliters of AP HRP solution to each covered slide. Add the formamide solution or one X PBS to keep the box moist but not soggy. Following that, incubate the slides in a humid box for 60 minutes at 37 degrees Celsius.
Wash the slides three times for five minutes in one X TBS supplemented with 0.05%TWEEN 20 on a rocker. After that, rinse the slides in DAP buffer for five minutes by leaving the plastic container on a rocker agitated gently. For chromogenic detection, add 100 microliters of NBT/BCIP substrate solution to each slide.
Carefully place the cover slips onto the slides, then incubate the slides in a humid box with substrate solution for 10 minutes to overnight at 37 degrees Celsius. Check the development by looking at the slides from time to time under the microscope. When the development is ready, stop the reaction by transferring the slides into water.
For TSA detection, use a syringe to move the HNPP/Fast Red substrate from the syringe filter, then add 100 microliters of HNPP/Fast Red substrate to each slide covered with a cover slip. After that, incubate the slides for 30 minutes with HNPP/Fast Red substrate at room temperature. After 30 minutes, remove the cover slips carefully and wash the slides three times for five minutes in one X TBS supplemented with 0.05%TWEEN 20.
Incubate the slides with 100 microliters of TSA substrate per covered slide for 10 minutes at room temperature. Remove the cover slips carefully and wash the slides three times for five minutes in one X TBS supplemented with 0.05%TWEEN 20 before embedding the slides in PBS glycerol. For chromogenic detection, observe the tissue sections using an optical microscope.
Shown here are the labeling pattern of LmigOR1 and LmigORco antisense probe on the consecutive sections of locust antenna. Here are the labeling pattern of LmigORco and LmigOR2 antisense probe on the consecutive sections of locust antenna. The illustration of the occasionally labeled dendritic-like structures are shown here.
For TSA detection, observe the tissue sections using a confocal microscope. Here, two color in situ hybridization was performed on the longitudinal antennal section to illustrate the expression of LmigOR1 and LmigORco. Localization of LmigOR1 expressing cells in cell clusters expressing LmigORco confirmed its neural identity, and here is a close view of the boxed areas.
Occasionally labeled dendritic-like structure is indicated by an arrow, and the circled areas indicate the olfactory receptor neurons cluster expressing LmigORco and sharing the same sensillum.
