I'm adding two coplaint protocols for different applications: I Abcam Troubleshooting form ?Immunohistochemistry- 1. Please describe the problem (high background, no staining etc). weak but overall, unspecific staining of the whole cell. No specific labelling, as it was seen before with previously purchased batch. Upon redistribution of protein (overexpression of GFP-tagged mutant construct), no redistribution of ab83 labelling, i.e. no co-localization of protein and antibody in IF. In Immuno-EM: overall staining of all cellular compartments in ultrathin cryosections 2. On what material are you testing the antibody in IHC? Cultured cell lines, primary cells ? Species? human ? Cell line? HelaP4, MT4, primary macrophages. ? Tissue? 3. How did you fix the samples? ? 3% Paraformaldehyde in PBS for fluorescence ? Other: Glutaraldehyde+PFA or PFA only for immuno-EM 4. Did you apply antigen retrieval step? NO ? Enzymatic method ? Heat mediated technique ? Other 5. How did you block the unspecific binding sites? 5% FCS in PBS/20mM glycine OR 5% goat serum in PBS/20mM glycine OR 1%fish skin gelatine/0.8% BSA in PBS/20mM glycine 6. Primary antibody ? Specification: ab83 Mouse monoclonal (4A10) AB to Tsg101, cross reacts with human and mouse. Lot 506 ? At what dilution(s) have you tested this antibody? 1:10- 1:300 ? Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)? Several attempts: 20-60 min incubation. Wash (PBS/glycine of PBS alone) 3-6 steps, 2-4 each, depending on number of wash steps. 7. Secondary antibody ? What secondary antibody are you using? IgGs: Donkey anti-mouse Cy3 Or donkey anti-mouse FITC for IF. For Immuno-EM: rabbit-anti-mouse IgG + ProteinA Gold ? Specification (in which species was it raised against)? See above ? At what dilution(s) have you tested this antibody? As established for the secondary abs in the lab (1:400 Cy3, 1:250 for FITC, 1:75 for rabbit-anti-mouse). ? Incubation, wash steps?. As for primary AB ? Do you know whether the problems you are experiencing come from the secondary?. Yes, I know. It's not the secondaries, since they work fine for all other applications. Negative controls (i.e. in this case incubation with secondary AB alone) are truly negative ? What detection method are you using? IF: fluorescence microscopy, wide-field and confocal. Electron microcopy: ProteinA Gold + uranyl acetate contrasting. 8. Background staining ? Please provide an image of your staining. See attachment "Tsg.ppt". 9. Which detection system did you use? 10. Did you apply positive and negative controls along with the samples? IF Negative control: secondary only (since Tsg101 is an endogenous protein). Additional control: redistribution of Tsg101 to internal aberrant endosomal structures (dn Vps4B overexpression). This leads to no difference in ab83 staining 11. Optimization attempts ? How many times have you tried the IHC? 3 ? Do you obtain the same results every time? yes ? What steps have you altered? AB dilution, blocking solution, incubation time II Abcam Troubleshooting form ?Immunohistochemistry- 1. Please describe the problem (high background, no staining etc). weak but overall, unspecific staining of the whole cell. No specific labelling, as it was seen before with previously purchased batch. Upon redistribution of protein (overexpression of GFP-tagged mutant construct), no redistribution of ab83 labelling, i.e. no co-localization of protein and antibody in IF. In Immuno-EM: overall staining of all cellular compartments in ultrathin cryosections 2. On what material are you testing the antibody in IHC? Cultured cell lines, primary cells ? Species? human ? Cell line? HelaP4, MT4, primary macrophages. ? Tissue? 3. How did you fix the samples? ? 3% Paraformaldehyde in PBS for fluorescence ? Other: Glutaraldehyde+PFA or PFA only for immuno-EM 4. Did you apply antigen retrieval step? NO ? Enzymatic method ? Heat mediated technique ? Other 5. How did you block the unspecific binding sites? 5% FCS in PBS/20mM glycine OR 5% goat serum in PBS/20mM glycine OR 1%fish skin gelatine/0.8% BSA in PBS/20mM glycine 6. Primary antibody ? Specification: ab83 Mouse monoclonal (4A10) AB to Tsg101, cross reacts with human and mouse. Lot 506 ? At what dilution(s) have you tested this antibody? 1:10- 1:300 ? Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)? Several attempts: 20-60 min incubation. Wash (PBS/glycine of PBS alone) 3-6 steps, 2-4 each, depending on number of wash steps. 7. Secondary antibody ? What secondary antibody are you using? IgGs: Donkey anti-mouse Cy3 Or donkey anti-mouse FITC for IF. For Immuno-EM: rabbit-anti-mouse IgG + ProteinA Gold ? Specification (in which species was it raised against)? See above ? At what dilution(s) have you tested this antibody? As established for the secondary abs in the lab (1:400 Cy3, 1:250 for FITC, 1:75 for rabbit-anti-mouse). ? Incubation, wash steps?. As for primary AB ? Do you know whether the problems you are experiencing come from the secondary?. Yes, I know. It's not the secondaries, since they work fine for all other applications. Negative controls (i.e. in this case incubation with secondary AB alone) are truly negative ? What detection method are you using? IF: fluorescence microscopy, wide-field and confocal. Electron microcopy: ProteinA Gold + uranyl acetate contrasting. 8. Background staining ? Please provide an image of your staining. See attachment "Tsg.ppt". 9. Which detection system did you use? 10. Did you apply positive and negative controls along with the samples? IF Negative control: secondary only (since Tsg101 is an endogenous protein). Additional control: redistribution of Tsg101 to internal aberrant endosomal structures (dn Vps4B overexpression). This leads to no difference in ab83 staining 11. Optimization attempts ? How many times have you tried the IHC? 3

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Asked on Nov 10 2004