13. Handling of the tissue as described post-fixation, without
cryofreezing or paraffin-embedding, greatly minimizes postprocessing. This preserves both tissue architecture and epitopes for immunofluorescence staining, and enables thick tissue imaging with even sections.
14. Primary and secondary antibodies for tissue staining are generally used at a dilution of 1:100 to 1:300, although the concentration used should be optimized as needed for each antibody.
DAPI is used at 1:500, and Alexa Fluor 647 conjugated phalloidin is used at 1:50.
15. As the tissue sections used are much thicker than standard
sections obtained from cryo- or paraffin-sectioning, care
should be taken to mount the samples in a way that does not
result in inadvertent compression of the samples. Inert vacuum
grease delivered via a 10 mL syringe provides a simple way for
mounting, as the vacuum grease can be used to create a “chamber” on the slide, where the mounting media and sample are
placed. A cover slip can then be gently and evenly applied, to
hold the sample in place. An alternative to the use of vacuum
grease is adhesive spacers available commercially from several
companies, such as Electron Microscopy Sciences (SecureSeal
imaging spacers) and SunJin Lab Co (iSpacer).
16. Decisions on the best channel acquisition settings to use reflect
a balance between the need to capture the widest possible
range of signal observed for a given reporter, which can span
log-scale magnitudes, and minimizing signal saturation. In
addition, it is critical to maintain GFP channel acquisition
settings across images for a given experimental set for each
transcriptional reporter, to allow comparison across images.
17. Images are generally restricted to 10–20 μm in depth as signal
loss becomes significant on a standard confocal microscope
with standard objectives at greater depths. If imaging deeper
into a tissue is desired, multiphoton confocal microscopy
and/or tissue optical clearing methods can be pursued [1, 36].
18. If red blood cell lysis is incomplete (indicated by presence of
red coloring in the cell pellet), resuspend the pellet again in
5 mL of ACK lysing buffer and repeat steps 8 and 9 in Subheading 3.6.
19. Antibodies for surface staining for flow cytometry are generally
used at a dilution of 1:200 to 1:500, although the concentration used should be optimized as needed for each antibody.
20. While samples can be stored at 4
C, protected from light, the
whole process of extraction of cells from the lung to imaging of
sorted samples should be completed within 1 week.
Exploiting Fluorescent Reporter M. tuberculosis Strains
381
cryofreezing or paraffin-embedding, greatly minimizes postprocessing. This preserves both tissue architecture and epitopes for immunofluorescence staining, and enables thick tissue imaging with even sections.
14. Primary and secondary antibodies for tissue staining are generally used at a dilution of 1:100 to 1:300, although the concentration used should be optimized as needed for each antibody.
DAPI is used at 1:500, and Alexa Fluor 647 conjugated phalloidin is used at 1:50.
15. As the tissue sections used are much thicker than standard
sections obtained from cryo- or paraffin-sectioning, care
should be taken to mount the samples in a way that does not
result in inadvertent compression of the samples. Inert vacuum
grease delivered via a 10 mL syringe provides a simple way for
mounting, as the vacuum grease can be used to create a “chamber” on the slide, where the mounting media and sample are
placed. A cover slip can then be gently and evenly applied, to
hold the sample in place. An alternative to the use of vacuum
grease is adhesive spacers available commercially from several
companies, such as Electron Microscopy Sciences (SecureSeal
imaging spacers) and SunJin Lab Co (iSpacer).
16. Decisions on the best channel acquisition settings to use reflect
a balance between the need to capture the widest possible
range of signal observed for a given reporter, which can span
log-scale magnitudes, and minimizing signal saturation. In
addition, it is critical to maintain GFP channel acquisition
settings across images for a given experimental set for each
transcriptional reporter, to allow comparison across images.
17. Images are generally restricted to 10–20 μm in depth as signal
loss becomes significant on a standard confocal microscope
with standard objectives at greater depths. If imaging deeper
into a tissue is desired, multiphoton confocal microscopy
and/or tissue optical clearing methods can be pursued [1, 36].
18. If red blood cell lysis is incomplete (indicated by presence of
red coloring in the cell pellet), resuspend the pellet again in
5 mL of ACK lysing buffer and repeat steps 8 and 9 in Subheading 3.6.
19. Antibodies for surface staining for flow cytometry are generally
used at a dilution of 1:200 to 1:500, although the concentration used should be optimized as needed for each antibody.
20. While samples can be stored at 4
C, protected from light, the
whole process of extraction of cells from the lung to imaging of
sorted samples should be completed within 1 week.
Exploiting Fluorescent Reporter M. tuberculosis Strains
381