-Take out the slides with the samples that were cut and kept at -80°C.
-Leave them to dry at RT, and once the glass is dry surround the sample with a small layer of ImmEdge Hydrophobic Barrier PAP Pen (Vector, H-400).
-Wait until the hydrophobic liquid is dry.
-Block the samples using 500uL of blocking medium (5% FBS in PBS+/+ plus 0.3% Triton 100x (Merck Millipore, 1122980101)) for 4 hours at RT.
-After that, add the primary antibodies diluted in the blocking medium.
-Iba1 1:500(Synaptic Systems, 234 004)
-GFAP 1:500 (Agilent Dako, Z033429-2)
-α-Actinin 1:500 (Sigma-Aldrich, A7811)
-Incubate the primary antibodies for 48 hours at 4°C.
-Then, wash four times with PBS+/+. First a quick wash, and then a 10 minute, 20 minutes and 30 minutes wash, at RT.
-Next, add the secondary antibodies diluted in blocking medium, and incubate for 2 hours at RT.
-AlexaFluor 488 Guinea Pig 1:500 (706-545-148) (Jackson Immuno Research)
-AlexaFluor 594 Rabbit 1:500 (711-585-152) (Jackson Immuno Research)
-AlexaFluor 647 Mouse 1:500 (715-695-151) (Jackson Immuno Research)
-After the secondary antibodies incubation, wash again with PBS-/- two times, one quick and 10 minutes.
-With the third wash, add the DAPI antibody, and incubate for 15 minutes at RT.
-Dapi 1:50.000 (Roth, 6335.1)
-Wash again with PBS-/- for 35 minutes at RT.
-Take away the wash, and leave the samples to dry a little bit.
-Mount the sections using VectaMount Permanent Mounting Medium (Biozol,
VEC-H-5000)and a glass coverslip (VWR, 24x50mm, 631-1574).
-Image the samples using a confocal microscope (Leica DMi8 Confocal Microscope) and software (Leica software).