Anti-Piccolo 抗体

• IHC-P

AbReview71457****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Piccolo antibody (AB20664)

Immunohistochemistry analysis (Formalin/PFA-fixed paraffin-embedded sections) of paraformaldehyde-fixed human brain tissue permeabilized with tritonX 0.05%. Stained with ab20664 at 1/300 dilution. Secondary antibody used was Alexa fluor® 488 goat anti-rabbit at 1/500 dilution. Blocking was done with 10% serum for 1 hour at 25°C. The sample was incubated with the primary antibody and 1% donkey serum for 1 hour at 25°C. Antigen retrieval method was heat mediated, Tris EDTA at 95°C 20mins.

This image is courtesy of an anonymous Abreview

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Piccolo antibody (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.

• IHC (PFA fixed)

Collaborator

Immunohistochemistry (PFA fixed) - Anti-Piccolo antibody (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.

This image is courtesy of Sophie Pezet, King's College London, United Kingdom

• WB

Ap25868****

Western blot - Anti-Piccolo antibody (AB20664)

The banding pattern shown in the image above is consistent with the literature which describes multiple bands >420 kDa as a result of proteolysis (PMID : 10707984).

This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes using lysates heated to 85°C before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab20664 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-Piccolo antibody (ab20664) at 1 µg/mL

Lane 1:

Brain (Rat) Tissue Lysate at 10 µg

Lane 2:

Cerebellum (Rat) Tissue Lysate at 10 µg

Lane 3:

Kidney (Rat) Tissue Lysate (Negative control) at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Predicted band size: 567 kDa

Observed band size: 200 kDa,460 kDa

true

Exposure time: 4min

• WB

Collaborator

Western blot - Anti-Piccolo antibody (AB20664)

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.

All lanes:

Western blot - Anti-Piccolo antibody (ab20664) at 0.2 µg/mL

Lane 1:

Rat brain lysate at 40 µg

Lane 2:

Mouse brain lysate at 50 µg

Secondary

All lanes:

Anti-rabbit HRP at 1/20000 dilution

Predicted band size: 567 kDa

Observed band size: 520 kDa,60 kDa

true

Exposure time: 10min

This image is courtesy of Randal Moldrich, CNRS UMR7637, ESPCI, France