Ab175470

Western blots were run by SDS-PAGE at 225 V for 39 min in Novex 4–20% Tris-glycine Mini Wedge 12-well gels (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) and transferred to nitrocellulose membrane (NCM) using iBlot 2 Dry Blotting System (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). NCMs were then blocked using 1× Blocker^TM fluorescent buffer (ThermoFisher Scientific, Waltham, MA, USA) for 1 h. Following blocking, 1:500 dilution of a primary antibody including EDA-Fn (SAB4200784; Millipore Sigma, Burlington, MA, USA), TSP-1 (ab85762; Abcam, Cambridge, MA, USA), and α-SMA (ab5694; Abcam, Cambridge, MA, USA), β-actin (ab184092; Abcam, Cambridge, MA, USA), vinculin (ab207440; Abcam, Cambridge, MA, USA), or vimentin (ab92547; Abcam, Cambridge, MA, USA) was added overnight at 4 °C. The NCMs were washed three times with Tris-Buffer Saline with 1% tween (TBST) for 5 min, followed by a 1 h incubation with the secondary antibody (1:2000); donkey anti-rabbit AlexaFluor 568 (ab175470; Abcam, Cambridge, MA, USA), at room temperature. The NCMs were imaged with the iBright FL 15,000 imaging system (ThermoFisher Scientific, Waltham, MA, USA) and analyzed with the iBright analysis software (ThermoFisher Scientific, Waltham, MA, USA).

Animals were sacrificed following resident-intruder or fear conditioning (see Results section for details about timing). Primary antibodies used were rabbit anti-phospho-ERK (Cell Signaling 9101, 1/500) and mouse anti-NeuN (Millipore MAB377, 1/200). Secondary antibodies were anti-rabbit-alexa-568 and anti-mouse-alexa-647 (Abcam ab175470 and ab150107, respectively, 1/1000).

The following primary antibodies were used with 1:200 concentrations: Anti-Phospho-CDK1 (Thr14, Tyr15) Mouse monoclonal Antibody (MABE229, EMD Millipore), Phospho-Histone H3 (Ser10) Polyclonal Antibody (PA5-17869, Thermo Scientific), Phospho-CHK1 (Ser345) Monoclonal Antibody (S.48.4) (MA5-15145, Thermo Scientific), Anti-alpha tubulin Mouse monoclonal [DM1A] antibody (ab7291, Abcam).
Secondary antibodies: Donkey Anti-Mouse IgG H&L (Alexa Fluor® 488) (Abcam, ab150105) (1:200), Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 568) (ab175470, Abcam) (1:200), Donkey Anti-Mouse IgG H&L (Alexa Fluor® 647) (ab150107, Abcam) (1:200), Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077, Abcam) (1:200). 0.5 μg/ml DAPI (diamidino-2-phenylindole) stain (62248, Thermo Scientific™) (1:200) was used to stain DNA.

Neural apoptosis was assessed by TUNEL staining via a DeadEnd Fluorometric TUNEL System (cat. # G3250, Promega Bio Sciences LLC, San Luis Obispo, CA, USA) The slides were incubated overnight with the rabbit anti-NeuN antibody (1:200, cat. # ab177487, Abcam). After probing with a relevant secondary antibody (1:200, cat. # ab175470, Abcam), a standard TUNEL procedure was performed as described previously [17 (link)]. Nuclei of cells were stained using 4′,6-diamidino-2-phenylindole (DAPI).

Electroporated brains were harvested at P2, fixed in 4% PFA overnight and thoroughly washed with 1 × PBS. The brains were embedded in 4% low-temperature melting agarose in 1 × PBS and cut into 60 µm thick sections on a Leica VT1000S Vibratome. The free-floating sections were permeabilized and blocked with blocking solution (10% FCS and 1% Triton X-100 in 1 × PBS) for 2 h at room temperature and stained in primary antibodies overnight at 4 °C. Primary antibodies, chicken anti-GFP (GFP-1020, AVES Labs) and rat anti-Ctip2 (ab18465, Abcam), were incubated 1:500 in blocking solution. The next day, sections were washed with 1 × PBS, and incubated in secondary antibodies: Alexa Fluor 488 Donkey anti-chicken (703–545-155, Jackson ImmunoResearch), Alexa Fluor 647 Donkey anti-rat (ab 150,155, Abcam), and Alexa Fluor 568 Donkey Anti-Rabbit IgG H&L (ab175470) 1:500 in blocking solution for 2 h at room temperature. Next, the sections were immersed in DAPI for 20 min, washed thoroughly with 1 × PBS, transferred onto microscopic glass slides, and mounted using Mowiol 4–88. Images were taken on an AxioScope A1 epifluorescence microscope.

Paraffin-embedded 4T1 tumor sections were dewaxed and antigen retrieval step performed in 10 mM citrate buffer in 95 °C water bath for 30 min. After 30 min at RT, slides were incubated in PBS-Tween 0.1%, 8% BSA for 1 h, then incubated overnight at 4 °C with primary antibodies: rat anti-GRP94 (LS-B3418-50) and rabbit anti-CD206 (LS-B9805-200, Clinisciences, Nanterre, France). Tissues were washed in PBS-Tween and incubated 1 h at RT with the following antibodies anti-rat Alexa 488 (Abcam, ab150153), anti-rabbit Alexa 568 (Abcam, ab175470). Microscopy images were acquired and analyzed as described for immunofluorescence.

Immunofluorescent staining was performed using goat polyclonal antibodies to c-Fos protein (1:200; Santa Cruz Biotechnology, sc-52-G), rabbit monoclonal antibodies (EPR4338) to hLf (1:150; Abcam, ab109000), and mouse monoclonal antibodies to NeuN (clone A60) (1:500; Millipore, MAB377) with subsequent detection with donkey anti-rabbit antibodies labeled with Alexa Fluor 488 fluorophore (1:500; Abcam, ab150129), donkey anti-rabbit antibodies labeled with Alexa Fluor 568 fluorophore (1:500; Abcam, ab175470), and donkey anti-mouse antibodies labeled with Alexa Fluor 647 fluorophore (1:500; Invitrogen, A-31571). Cell nuclei were poststained with Hoechst 33258 (1:1000; Invitrogen, H-1398). Stained cultures were digitized and analyzed (see Section 4.2). The number of c-Fos-positive nuclei was expressed as a percentage of the total number of nuclei [22 (link)]; the arithmetic mean was calculated for 10 fields of view in each culture.