Wash with Tris several times for a total of 15 min.
Wash with filtered ultrapure water: wash the sections once, and then wash the
whole coverslip. Note: At this point it is very easy for the sections to dry out, so
be careful to always leave some water behind.
Mount with SlowFade Gold antifade reagent with DAPI (Invitrogen). The DAPI
stain helps find the sections and focus. For mounting: remove some, but not all of
the water from the array, then add a couple of drops of mounting medium and
remove more of the water.
6. Elution
6A. Required materials:
NaOH (10 N)
SDS (20% w/v; Roche 1666924)
Tris buffer
6B. Preparations:
Elution solution (0.2 M NaOH and 0.02% SDS in ultrapure water): Add 200 ll
of NaOH (10 N) and 10 ll SDS to 10 ml of ultrapure water. Store at room
temperature for up to 6 months.
6C. Procedures:
Add water around the edge of the coverslip to help detach it from the microscope
slide. Wait *1 min. The coverslip will float up. Pick it up with tweezers and
wash away mounting medium with ultrapure water. Note: It is best to perform
this step as soon as finished with imaging. Leaving the sections too long with the
mounting medium will damage the sections.
Apply the elution solution for 20 min at room temperature (add the solution
gently to the sections, do not wash with the elution solution). Elution time may
vary for different antibodies; can be tested by applying only the secondary
antibody after elution and checking for remaining fluorescence. Some antibodies
cannot be sufficiently eluted, for example GABA, and such antibodies are best
left for the last cycle of immunostaining.
Wash with Tris buffer (15 min). The initial wash should be slow.
A new immunostaining can be performed following the same procedure as
described in Sect. 4.
Alternatively, to store the coverslip for possible staining at a later time, after the
15 min Tris wash briefly rinse with filtered ultrapure water. After the sections
have dried, the coverslips can be stored for a long period of time.
6 Conjugate Immunofluorescence—SEM Array Tomography …
177
Wash with filtered ultrapure water: wash the sections once, and then wash the
whole coverslip. Note: At this point it is very easy for the sections to dry out, so
be careful to always leave some water behind.
Mount with SlowFade Gold antifade reagent with DAPI (Invitrogen). The DAPI
stain helps find the sections and focus. For mounting: remove some, but not all of
the water from the array, then add a couple of drops of mounting medium and
remove more of the water.
6. Elution
6A. Required materials:
NaOH (10 N)
SDS (20% w/v; Roche 1666924)
Tris buffer
6B. Preparations:
Elution solution (0.2 M NaOH and 0.02% SDS in ultrapure water): Add 200 ll
of NaOH (10 N) and 10 ll SDS to 10 ml of ultrapure water. Store at room
temperature for up to 6 months.
6C. Procedures:
Add water around the edge of the coverslip to help detach it from the microscope
slide. Wait *1 min. The coverslip will float up. Pick it up with tweezers and
wash away mounting medium with ultrapure water. Note: It is best to perform
this step as soon as finished with imaging. Leaving the sections too long with the
mounting medium will damage the sections.
Apply the elution solution for 20 min at room temperature (add the solution
gently to the sections, do not wash with the elution solution). Elution time may
vary for different antibodies; can be tested by applying only the secondary
antibody after elution and checking for remaining fluorescence. Some antibodies
cannot be sufficiently eluted, for example GABA, and such antibodies are best
left for the last cycle of immunostaining.
Wash with Tris buffer (15 min). The initial wash should be slow.
A new immunostaining can be performed following the same procedure as
described in Sect. 4.
Alternatively, to store the coverslip for possible staining at a later time, after the
15 min Tris wash briefly rinse with filtered ultrapure water. After the sections
have dried, the coverslips can be stored for a long period of time.
6 Conjugate Immunofluorescence—SEM Array Tomography …
177