21. Tissue is transferred into a drop of the desired mounting
solution and the root tips are separated under a stereomicroscope. To prevent tissue crushing when covering the tissue
sample with a coverslip, we use a drop of clear nail polish on
the glass slide at the edges of the cover glass as spacer.
22. Do not use ethanol to stop the reaction or clearing solution like
for NBT/BCIP staining procedure. The precipitated Fast Red
is washed out by ethanol and bleached by clearing solution.
Thus, it is not possible to clear the tissue for optimizing DIC
microscopy images.
23. For the Fast Red dye, we used a 561 nm laser for excitation and
the emission was detected between 570 and 640 nm. The
DNA was counterstained with DAPI, which was excited at
405 nm and the emitted fluorescence was detected in the
range of 420–470 nm.
24. The TSA staining reaction leads to the multi-labeling of the
antibody and the enzyme itself, leading to its inactivation.
Prolonged staining reactions are useless and will only increase
the background.
25. For the TSA-CY3 staining, we used a 561 nm laser for excitation and the emission was detected between 570 and 640 nm.
For the TSA-FITC staining, we used a 488 nm laser for excitation and the emission was detected between 500 and 550 nm.
The DNA was counterstained with DAPI, which was excited at
405 nm and the emitted fluorescence was detected between
420 and 470 nm.
26. Try both labeling orientations: probe 1 labeled with DIG and
probe 2 with FITC and vice versa, as the probes might have
different efficiencies.
27. You may first apply the anti-digoxigenin-HRP-conjugated
antibody and perform the staining reaction before applying
the anti-fluorescein-HRP-conjugated antibody and the second
staining reaction. However, to optimize the labeling, also try to
do it the other way around.
Acknowledgments
This study was supported by the Collaborate Research Center
SFB960 of the German Research Foundation (DFG—Germany)
with funding to T.D.
20
Thomas Dresselhaus and Andrea Bleckmann
solution and the root tips are separated under a stereomicroscope. To prevent tissue crushing when covering the tissue
sample with a coverslip, we use a drop of clear nail polish on
the glass slide at the edges of the cover glass as spacer.
22. Do not use ethanol to stop the reaction or clearing solution like
for NBT/BCIP staining procedure. The precipitated Fast Red
is washed out by ethanol and bleached by clearing solution.
Thus, it is not possible to clear the tissue for optimizing DIC
microscopy images.
23. For the Fast Red dye, we used a 561 nm laser for excitation and
the emission was detected between 570 and 640 nm. The
DNA was counterstained with DAPI, which was excited at
405 nm and the emitted fluorescence was detected in the
range of 420–470 nm.
24. The TSA staining reaction leads to the multi-labeling of the
antibody and the enzyme itself, leading to its inactivation.
Prolonged staining reactions are useless and will only increase
the background.
25. For the TSA-CY3 staining, we used a 561 nm laser for excitation and the emission was detected between 570 and 640 nm.
For the TSA-FITC staining, we used a 488 nm laser for excitation and the emission was detected between 500 and 550 nm.
The DNA was counterstained with DAPI, which was excited at
405 nm and the emitted fluorescence was detected between
420 and 470 nm.
26. Try both labeling orientations: probe 1 labeled with DIG and
probe 2 with FITC and vice versa, as the probes might have
different efficiencies.
27. You may first apply the anti-digoxigenin-HRP-conjugated
antibody and perform the staining reaction before applying
the anti-fluorescein-HRP-conjugated antibody and the second
staining reaction. However, to optimize the labeling, also try to
do it the other way around.
Acknowledgments
This study was supported by the Collaborate Research Center
SFB960 of the German Research Foundation (DFG—Germany)
with funding to T.D.
20
Thomas Dresselhaus and Andrea Bleckmann