Bovine serum albumin (e.g. AURION BSA-C, Electron Microscopy
Sciences 25557)
Tween-20 (Electron Microscopy Sciences 25564)
Primary antibodies
Secondary antibodies: the appropriate species of Alexa Fluor 488, 594 and
647, IgG (H + L), highly cross-adsorbed (Invitrogen)
Transfer pipets, extra-fine tip polyethylene (Fisher Scientific 13-711-31)
Mounting medium: SlowFade Gold antifade reagent with DAPI
(Invitrogen)
Glass Slides, precleaned Gold Seal Rite-On micro slides (Fisher Scientific
12-518-103)
5B. Preparations:
Tris buffer: dissolve 1 tablet in 15 ml of ultrapure water
1% Na borohydride: 10 mg of Na borohydride in 1 ml of Tris. Prepare
immediately before use. Do not close the Eppendorf tube or it may burst from the
building pressure.
50 mM Glycine: 4 mg glycine in 1 ml of Tris
Blocking solution: Make a 1% stock of Tween-20 (10 ll Tween in 1 ml of
ultrapure water). Then add 50 ll of the 1% Tween stock solution to 0.94 ml Tris.
Add 10 ll of Aurion BSA.
5C. Procedures:
Encircle the sections with a PAP pen leaving some extra space at the two ends of
the ribbon. Put the coverslips in a Petri dish or box, and add wet KimWipes on
the side to prevent evaporation of solutions. Keep closed during incubation
times. All incubations except primary antibodies are done at room temperature.
Cover the sections with 1% Na borohydride for 3 min. Remove big bubbles that
form during the incubation by gently tapping the box to the bench. Wash with Tris
for a total of 20 min. Washing of the sections is done in several periods (3–4 periods
of about 10–15 s) of continuous flow of buffer, by adding buffer with a plastic
transfer pipet on one side of the sections and removing buffer with another pipet (or
a pipet tip connected to a vacuum line) on the opposite side of the sections.
Remove Tris and add 50 mM glycine for *5 min.
Remove glycine and apply blocking solution for *5 min (no need to wash).
Remove blocking solution and apply primary antibody in Blocking Solution for
2 h at room temperature or overnight at 4 °C (no need to wash). Spin down the
antibody solution at 13,000 rpm for 2 min before applying to sections.
Note: After this step, it is important not to let the sections dry at any point!
Wash several times with Tris for a total of 15 min.
Apply secondary antibody (e.g. Alexa, 1:150) in blocking solution for 30 min.
Spin down the antibody solution at 13,000 rpm for 2 min before applying to
sections. Keep in the dark.
176
K. D. Micheva and K. D. Phend
Sciences 25557)
Tween-20 (Electron Microscopy Sciences 25564)
Primary antibodies
Secondary antibodies: the appropriate species of Alexa Fluor 488, 594 and
647, IgG (H + L), highly cross-adsorbed (Invitrogen)
Transfer pipets, extra-fine tip polyethylene (Fisher Scientific 13-711-31)
Mounting medium: SlowFade Gold antifade reagent with DAPI
(Invitrogen)
Glass Slides, precleaned Gold Seal Rite-On micro slides (Fisher Scientific
12-518-103)
5B. Preparations:
Tris buffer: dissolve 1 tablet in 15 ml of ultrapure water
1% Na borohydride: 10 mg of Na borohydride in 1 ml of Tris. Prepare
immediately before use. Do not close the Eppendorf tube or it may burst from the
building pressure.
50 mM Glycine: 4 mg glycine in 1 ml of Tris
Blocking solution: Make a 1% stock of Tween-20 (10 ll Tween in 1 ml of
ultrapure water). Then add 50 ll of the 1% Tween stock solution to 0.94 ml Tris.
Add 10 ll of Aurion BSA.
5C. Procedures:
Encircle the sections with a PAP pen leaving some extra space at the two ends of
the ribbon. Put the coverslips in a Petri dish or box, and add wet KimWipes on
the side to prevent evaporation of solutions. Keep closed during incubation
times. All incubations except primary antibodies are done at room temperature.
Cover the sections with 1% Na borohydride for 3 min. Remove big bubbles that
form during the incubation by gently tapping the box to the bench. Wash with Tris
for a total of 20 min. Washing of the sections is done in several periods (3–4 periods
of about 10–15 s) of continuous flow of buffer, by adding buffer with a plastic
transfer pipet on one side of the sections and removing buffer with another pipet (or
a pipet tip connected to a vacuum line) on the opposite side of the sections.
Remove Tris and add 50 mM glycine for *5 min.
Remove glycine and apply blocking solution for *5 min (no need to wash).
Remove blocking solution and apply primary antibody in Blocking Solution for
2 h at room temperature or overnight at 4 °C (no need to wash). Spin down the
antibody solution at 13,000 rpm for 2 min before applying to sections.
Note: After this step, it is important not to let the sections dry at any point!
Wash several times with Tris for a total of 15 min.
Apply secondary antibody (e.g. Alexa, 1:150) in blocking solution for 30 min.
Spin down the antibody solution at 13,000 rpm for 2 min before applying to
sections. Keep in the dark.
176
K. D. Micheva and K. D. Phend