Anti-Piccolo antibody - Synaptic Marker (ab20664)

Western blot - Piccolo antibody - Synaptic Marker (ab20664)

All lanes : Anti-Piccolo antibody - Synaptic Marker (ab20664) at 0.2 µg/ml

Lane 1 : Rat brain lysate at 40 µg
Lane 2 : Mouse brain lysate at 50 µg

Secondary
Anti-rabbit HRP at 1/20000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 520 kDa
Observed band size : 520 kDa
Additional bands at : 60 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 10 minutes

Primary Antibody : anti-Piccolo (ab20664) for 1h in 5% Whole Milk Powder (WMP); Secondary Antibody : Anti-rabbit HRP (1/20000) for 1h in 5% WMP; Migration medium: Laemmeli + glycerol + 5% B-mercaptoethanol; Transfer : Tris/Glycine, 20% ethanol

NB: Gels higher than 8% acrylamide were tried without success; 8% or lower is recommended. Denaturing of samples at 70C or 95C was not successful. Reducing conditions were used and 20% ethanol was employed for the nitrocellulose membrane transfer. All steps performed at RT.

Randal Moldrich, CNRS UMR7637, ESPCI, France

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Piccolo antibody - Synaptic Marker (ab20664)

IHC image of Piccolo staining in rat brain FFPE section, performed on a Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab20664, 5µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

Western blot - Piccolo antibody - Synaptic Marker (ab20664)

All lanes : Anti-Piccolo antibody - Synaptic Marker (ab20664) at 1 µg/ml

Lane 1 : Brain (Rat) Tissue Lysate
Lane 2 : Brain (Mouse) Tissue Lysate
Lane 3 : Brain (Rat) Tissue Lysate with Piccolo peptide (ab23400) at 1 µg/ml
Lane 4 : Brain (Mouse) Tissue Lysate with Piccolo peptide (ab23400) at 1 µg/ml

Lysates/proteins at 30 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size : 520 kDa
Observed band size : 275 kDa (why is the actual band size different from the predicted?)
Additional bands at : 31 kDa,60 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 4 minutes

Immunohistochemistry (PFA fixed) - Piccolo antibody - Synaptic Marker (ab20664)

Immuofluorescent staining for Piccolo ab20664 in [A] rat brain hippocampus (X20 objective) and [B] rat brain cortex (X40 objective). Tissue preparation: rat brain tissue was perfusion fixed (4% PFA) followed by post fix and cryoprotection in 20% sucrose before freezing in OCT. 30µm coronal sections were cut on a cryostat for free floating IHC. Primary antibody ab20664 was used at 1/100 (5µg/ml) incubated overnight at room temperature in PBST (triton 0.3%). Secondary antibody used: anti-rabbit Alexa fluor 488 (1/1000) incubated for 2 hours at room temperature.

Sophie Pezet, King`s college London, United Kingdom