Immunohistochemistry

After imaging and photoconversion in the 2p microscope, slices were fixed in a PBS solution containing 4% PFA for 30 min at room temperature (RT), washed and kept overnight in PBS at 4°C. Slices were then incubated in blocking buffer (1x PBS, 0.3% TritonX, 5% goat serum) for 2 h at RT. Next, sections were placed in the primary antibody (1:1,000, mouse Anti-CaMPARI-Red 4F6-1 clone, a gift from Eric Schreiter) carrier solution (PBS, 0.3% TritonX, 1% goat serum, 1% BSA) and incubated overnight at 4°C. After 3x washing with 1xPBS for 5 min, the sections were incubated for 2 h at RT in the secondary antibody (1:1,000, goat anti mouse Alexa Fluor 647, Life Technologies, United States) carrier solution (1x PBS, 0.3% TritonX, 5% goat serum). Sections were washed 3 × 10 min in 1x PBS, mounted on coverslips using Immu-Mount (Shandon, United States), and imaged with a confocal microscope (Olympus FV 1000) using a 20x oil immersion objective (UPLSAPO 20X NA 0.85). Two-channel images were obtained at 1024 × 1024 pixel resolution. Excitation/emission spectra and filters were selected using the automatic dye selection function of the Olympus FluoView software (Alexa 488 and Alexa 647).