Anti-LAMP1 antibody [LY1C6] - Lysosome Marker (ab13523)

Dear Supplier,

Please provide the quote for the item as below:

1) Anti-LAMP-1 antibody[LY1C6]-Lysosome Marker (ab 13523)

By the way, I want to stain lysosome in red color. Whether does your product contain a red dye. May I know if I need to purchase another Alexa Fluor568 conjugated goat anti mouse?

Thanks and hope to hear from you soon.

Research Fellow

ANSWER:

Thank you for your enquiry and your interest in our products.

I believe my colleague has already provided a quote for you.

Regarding the immunostaining - ab13523 is an unconjugated primary antibody so you would need a compatible secondary antibody for the detection. The isotype of this antibody is IgG1 and it is raised in mouse, thus it is necessary to apply a secondary antibody which recognizes mouse IgG1 and labelled with a fluorochrome.

Currently, we do not have Alexa Fluor conjugated secondary antibodies in our catalogue, however we do stock DyLight and Chromeo conjugated-labelled secondaries (new family of dyes) which have several advantages:

- Bright fluorescence – intense emission provides superior sensitivity

- Narrow emission spectra – negligible bleed-through between fluorochrome channels makes multi-color imaging easy

- Highly photostable – better resistance to photobleaching

- Completely stable in buffers ranging from pH 4-9

- Instrument compatibility – DyLight® can be visualized with common laser and filter settings

Some further information is available at this site:

http://www.abcam.com/index.html?pageconfig=resource&rid=12934

You could conduct your search for suitable secondary antibody using our advanced search engine at this site:

http://www.abcam.com/index.html?pageconfig=productmap&cl=918

I hope this helps and if I can assist further, please do not hesitate to contact me.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 170028 DESCRIPTION OF THE PROBLEM No staining SAMPLE Baby Hampster Kidney cells (BHK-21). PRIMARY ANTIBODY Mouse monoclonal Lysosome Marker (ab13523) at various dilutions. 1:200, 1:100, 1:50. DETECTION METHOD Inverted Epifluorescence microscopes (Nikon and Zeiss) POSITIVE AND NEGATIVE CONTROLS USED Used abcam mouse anti-PDI anitbody (ab2792-100) in parallel. This antibody has been excellent for me in the past and was used as a postive control for my proceedure and seconday antibody. Postive ER staining in these cells, but absolutely no staining in the Lamp-1 stained cells. ANTIBODY STORAGE CONDITIONS Aliquoted anitbody and stored at -20 deg C. FIXATION OF SAMPLE 100% Methanol for 15 mins. at -20 deg C. ANTIGEN RETRIEVAL n/a PERMEABILIZATION STEP 100% Methanol BLOCKING CONDITIONS PBS + 0.2% gelatin overnight at 4 deg C. SECONDARY ANTIBODY Donkey anti-Mouse Alexa Fluor 488 (Molecular Probes) at 1:200 dilution HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Changed dilutions to as low as 1:50. ADDITIONAL NOTES I really like abcam antibodies, I have several organelle markers that I use on a regular basis. However, I am very disappointed with this antibody. I expected it to be the same quality as my others. I need a mouse antibody to Lamp-1 or another lysosome resident protein if you have any suggestions.

ANSWER:

I think your protocol is very good and very similar to the one I have used for ICC, and have been investigating what successful methods were used with ab13523 in ICC. I conclude that I think the problem lies in the fixation step and would like to suggest trying a different fixative. It may be that simply fixing with methanol but for 5 minutes only will give you better results (in our experience 15 min can be overfixing), but the literature seems to recommend a paraformaldehyde based fixative as best to use.

For example, Harter C & Mellman I Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane. J Cell Biol 117:311-25 (1992) said "To monitor internalization by immunofluorescence, living cells grown on glass Coverslips were incubated with hybridoma culture supernatant containing the mouse monoclonal anti-rat 1gp120 antibody Ly1C6 IgG (Lewis et al ., 1985) for 2 h on ice or at 37°C . Cells incubated on ice were washed three times with PBS containing 10% goat serum to remove excess antibody before warming to 37°C for 30 min or 1 h to allow internalization . Cells were then fixed with paraformaldehyde-lysine-periodate (McLean and Nakane, 1974) for 1 h at room temperature after permeabilization with 0.01% saponin in PBS for 7 min and labeling with an affinity-purified, Texas red-conjugated goat anti-mouse IgG ... After extensive washing with PBS containing 10%goat serum, cells were mounted in Mowiol and viewed with an Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY)."

Isobe I et al. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) causes Akt phosphorylation and morphological changes in intracellular organellae in cultured rat astrocytes. J Neurochem 77:274-80 (2001) said "Cultures grown on chamber slides were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min. After washing three times with PBS, the cells were permeabilized with ethanol containing 5% acetic acid for 5 min at - 20°C. The cells were again washed three times with PBS and incubated in PBS containing 5% normal goat serum (NGS) for 1 h at room temperature, and thereafter with primary antibodies at 4°C overnight...and mouse monoclonal antibody against Lamp1 .. were used at 1 :100 dilution in PBS containing 5% NGS. Cultures incubated without primary antibodies were used as the negative controls. After washing three times with PBS, the samples were incubated with secondary antibodies for 1 h at room temperature. The secondary antibodies, ...FITC-labeled goat anti-mouse IgG ...were used at 1 : 100 dilution in PBS containing 5% NGS. The cells were washed three times with PBS, mounted in a mounting medium (Vectashield; Vector Laboratories, Burlingame, CA, USA)".

I hope this information will be useful. Please do not hesitate to contact me if you still experience problems and I can offer you a replacement vial or refund if the antibody was purchased in the last 90 days,

DESCRIPTION OF THE PROBLEM No staining

SAMPLE CELL TYPES - PC3 AND LnCAP.

PRIMARY ANTIBODY 1:100 dilution of anti-LAMP-1 ab13523 for 1 hr. and 3 times washing with PBS

SECONDARY ANTIBODY 2 ug/ml of Goat anti-rabbit (IgGs) - Molecular Probes- 1hr and later 3 times washing with PBS, 5 min intervals.

DETECTION METHOD Using LSM confocal microscope.

POSITIVE AND NEGATIVE CONTROLS USED No antibody control and for checking if the secondary antibody works or not,another antibody was used and it works fine.

ANTIBODY STORAGE CONDITIONS STORE AT REFRIGERATOR

FIXATION OF SAMPLE Paraformaldehyde- 3.7% for 20 min

ANTIGEN RETRIEVAL No

PERMEABILIZATION STEP 0.1 % triton X-100 for 10 min

BLOCKING CONDITIONS 10% Normal goat serum diluted in PBS for 30 min.

WHAT STEPS HAVE YOU ALTERED? First I used 1: 250 of antibody but later i used 1:100 antibody.

ANSWER:

Thank you for your enquiry and for providing more information about your protocol.

This antibody cross-reacts with rat and hamster LAMP1 but it has not been tested in other species. Unfortunately, we do not have any data indicating its suitability in huma samples.

As we understand you used two human cell lines; PC3 and LnCAP are both human prostatic carcinoma cell lines. We would strongly suggest staining rat liver tissue section as positive control.

You could also need to incubate the samples with the primary antibody for longer time (i.e. for 2 hrs) to see if the signal is better or not.

It may also worth checking other fixatives - ice cold acetone, ethanol or methanol.

We hope this will help.