Rabbit anti dsred antibody

We used the HEK293T cell system^{70 (link),75 (link)} to evaluate the efficacy of CNTNAP2 or AHI1 knockdown. HEK293T cells in a 24-well dish were transfected with an mOrange2-fused cDNA (CNTNAP2-mOrange or AHI1-mOrange) together with one of the following vector, an RNAi-knockdown (CNTNAP2-knockdown or AHI1-knockdown) vector, a rescue (CNTNAP2-rescue or AHI1-rescue) vector, or a scramble (CNTNAP2-Scr or AHI1-Scr) vector, using X-tremeGENE 9 reagents. One day later, these cells were fixed. After permeabilization by Triton-X-100 and blocking, primary antibodies (a Rat anti-GFP antibody, Nacalai Tesque; a Rabbit anti-DsRed antibody, Clontech laboratories inc.) were applied. Fluorescence signals from these cells were examined under a confocal laser scanning microscope (FV1200, Olympus).

Following recording, slices were placed in a fixative solution of 4% paraformaldehyde in PB for at least 24 h. The sections were then rinsed in PB and incubated overnight in a 1:1000 dilution of streptavidin conjugated to AlexaFluor-633 (Invitrogen) in PB containing 1% Triton X-100. The following day, the slices were washed in PB, preincubated in 10% normal goat serum in PB, and then incubated overnight in a 1:500 dilution of a rabbit anti-DSred antibody (Clontech, catalog #632496) in PB with 1% normal goat serum. The following day, the sections were rinsed in PB and incubated for 1 h in a 1:100 dilution of a goat-anti-rabbit antibody conjugated to AlexaFluor-546 (Invitrogen). The sections were then rinsed in PB and mounted on slides to be imaged with a confocal microscope (Olympus FV1200BX61).

Rats were deeply anesthetized with Nembutal and perfused transcardially with PBS followed by 4% paraformaldehyde (PFA). Brains were removed and postfixed in 4% PFA overnight, and then placed into 30% sucrose in 0.1 M phosphate buffer. Brains were then sectioned at 40 μm using a cryostat, and stored in cryoprotectant. To visualize viral expression, free‐floating coronal sections were washed two times in 1X PBS for 10 min and then blocked for 1–2 hr at room temperature in a solution of 5% normal goat serum and 1% Triton X‐100 dissolved in PBS. Sections were then washed three times in PBS for 15 min and incubated in blocking solution containing rabbit anti‐DsRed antibody (1:1000; Clontech, Mountain View, CA) with gentle agitation at 4°C for 18–22 hr. Sections were rinsed three times in the blocking solution and incubated in AlexaFluor‐594‐conjugated (red) goat secondary antibody (1:500; Invitrogen, Carlsbad, CA) for 2 hr. Sections were then washed three times in PBS for 30 min, mounted on slides, and coverslipped with ProLong Gold mounting medium with DAPI. All images were acquired using a Keyence (BZ‐X710) microscope with a ×4 or ×20 objective (CFI Plan Apo), CCD camera, and the BZ‐X analyzer software.

All fluorescent images were obtained with a Zeiss LSM710 confocal microscope, using a ×20 or x40 objective lens. The following antibodies were used: rabbit anti-dsRed antibody (1:50 from Clontech or 1:500 from Dr. S.W. Kang), rabbit anti-GFP (1:2000, Molecular Probes, catalogue no. A6455), mouse anti-armadillo (1:500; Developmental Studies Hybridoma Bank, N2 7A1, University of Iowa, USA), monoclonal anti-rhodopsin1 (1:500; Developmental Studies Hybridoma Bank, 4C5, University of Iowa, USA), actin-phalloidin (1:200; Molecular Probes). To generate the guinea-pig anti-ATF4 antibody, the full length of the ATF4 coding sequence was subcloned into XhoI and NotI sites in pET14b (Novagen). The resulting ~50 kDa His-tagged recombinant protein was purified to generate a polyclonal antibody. The antisera were subsequently affinity purified against the same epitope [19 (link)].

Adeno-associated virus (AAV) designer receptors exclusively activated by designer drug (DREADD) vectors (Addgene) were stereotaxically infused in the ventromedial PFC (including the central part of the PrL and IL cortices) of male PV:Cre mice. Briefly, AAV2/hSyn-DIO-hm3D(Gq)-mCherry (“hM3DGq”) was injected bilaterally (0.5 μl/side, ∼10¹² viral genomes/ml) into mPFC using the following coordinates: anteroposterior, +1.7 mm; mediolateral, ±0.2 mm; dorsoventral, −2.6 mm. AAV2/hSyn-DIO-mCherry was used as the control virus. Previous work demonstrated that chemogenetic activation of PV⁺ neurons from both PrL and IL regions increases anxiety-like behaviors in female mice (Page et al., 2019 (link)). To be able to compare our findings with this study, we opted to use a similar strategy. Mice remained undisturbed for at least 21 d after surgery to allow full expression of the DREADD virus in PV⁺ cells before the commencement of behavioral testing. All mice received a daily intraperitoneal injection of clozapine-N-oxide (CNO; 0.5 mg/kg) 30 min before daily handling or stressor throughout the UCMS period. After the completion of behavioral assays, viral injection sites were verified using a rabbit anti-DsRed antibody (1:1000; TaKaRa Bio) followed by an Alexa Fluor anti-rabbit 555 secondary antibody (1:500; Thermo Fisher Scientific) to target mCherry in prefrontal brain sections.

Staining of pERK was performed as previously described^{18 (link)} using rabbit monoclonal anti-pERK antibody (4376; Cell Signaling Technology, Beverly, MA, USA) at a 100-fold dilution. Other staining experiments were also performed as previously described^{18 (link)}. The following primary antibodies were used: rabbit anti-CoRest antibody at a 100-fold dilution; rabbit monoclonal anti-myc antibody (2278; Cell Signaling Technology, Beverly, MA, USA) at a 100-fold dilution; mouse nc82 antibody (Developmental Studies Hybridoma Bank, Univ. Iowa, USA) for the detection of the presynaptic protein Bruchpilot at a 50-fold dilution; rabbit anti-DsRed antibody (632496; Takara, Shiga, Japan) at a 200-fold dilution; and chicken anti-GFP antibody (ab13970; Abcam, Cambridge, MA, USA) at a 2,000-fold dilution. The following secondary antibodies were used at 500-fold dilutions: donkey anti-chicken immunoglobulin Y (IgY) Alexa 488 antibody (703-545-155; Jackson ImmunoResearch Labs, Inc., West Grove, PA, USA), donkey anti-mouse IgG Alexa Fluor 488 antibody (715-545-150; Jackson ImmunoResearch Labs, Inc., West Grove, PA, USA), and donkey anti-rabbit IgG Cy3 antibody (711-166-152; Jackson ImmunoResearch Labs, Inc., West Grove, PA, USA). Images were captured using a confocal microscope LSM780 (Zeiss Microsystems, Jena, Germany) or FV1000 (Olympus, Tokyo, Japan).