Dylight594 goat anti rabbit

The enucleated eyes from each mouse were fixed in 4% paraformaldehyde overnight; the corneas were removed by cutting in a circular path along the ora serrata with small scissors, holding the eye at the limbus with forceps. The lenses were pulled out with forceps. Vitreous humor was removed as much as possible, and the retina was detached from the eyecup by positioning forceps between the retina and the eyecup, moving the forceps slowly around the circumference. After separation, the retina was permeated by freezing in 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 15 min at −70 °C. The free-floating retinae were then washed in 0.5% Triton PBS three times for 10 min each and incubated with primary rabbit anti-RBPMS (RNA binding protein with multiple splicing) antibody (1:500, rabbit polyclonal, Abcam, cat. No. ab194213) overnight at 4 °C. Retinae were then washed three times for 10 min each with PBS and incubated with secondary antibody (Dylight594 Goat anti-rabbit 1: 1000, Thermo Fisher Scientific, cat No. A11008) at room temperature for 2 h. Retinae were then thoroughly washed in PBS and mounted with the vitreal side up on slides. To mount the entire retina flat on the slide, incisions were made from the periphery towards the center and then were cover-slipped with Vectashield anti-fade mounting medium solution with DAPI (Vector labs, Burlingame, Ca, USA).

Mice were anesthetized using 1.25% Avertin, saline and 4% paraformaldehyde (PFA) were injected into the heart. The brain tissues were fixed in PFA and were dehydrated in 30% sucrose solution for 48 h. Brain tissue was sectioned into coronal sections containing BLA or mPFC (30 μm thick) using a slide microtome (SM2010R, Leica, Germany). After PBS washing and BSA blocking, the brain slices were incubated with a primary antibody (Rabbit Anti-CaMKIIa, #ab_305050, Abcam; 1:500) at 4 °C for 48 h, followed by a secondary antibody (DyLight 594 Goat anti-rabbit; # AB_36933, Thermo Fisher Scientific, 1:500). Using a confocal microscope (ZEISS, Germany) to capture confocal immunofluorescence images and the fluorescent intensity was analyzed using ImageJ.