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IEM Protocol:white cells damaged by arsenic exposure.

Edit IEM Protocol:white cells damaged by arsenic exposure. here.
IEM Protocol: Whole cells pellet from Suti
Roger D. Sloboda(biology) and Sutisak Kitareewan(Pharm/tox) in IEM of white cells damaged by arsenic exposure to provide an insight on alterations to the lysosomal bodies and the proteins within these lysosomes.

1. Fix cells in 4% paraformaldehyde (PF)/ 1%GTA in PBS for 2 hours, RT. Western blot test shows IEM label still good with 1%GTA added.

2. Rinse in PBS for 15 min.

3. Quench free aldehydes with 50mM glycine in PBS

4. Dehydration/Infiltration/Polymerization: (Newman pp. 55-65; pp. 87-99). Dehydrate: 50% ETOH - 5 min and 70%ETOH - 2 changes, 5 min each

5. LR White: 85%ETOH - 2:1 2 changes 30 min each Error: this should not happen

6. LR White - 3 changes 30-60 min each, on a gentle Rotamix.

7. Overnight in fridge (@4oC), on a gentle Rotamix. Or got right to step 8.

8. LR white - 1 change 1 hour at RT

9. Place tissue in gelatin capsules, add fresh LR White; seal capsules; allow to settle for 1 hour.

10. Polymerize at 50oC for 24 hours.

NOTE: if you need to centrifuge material and you have a small sample size, place cells in trimmed beam capsule, seal and centrifuge inside a 12-15ml centrifuge tube {with a cotton bottom pad}, using a clinical centrifuge. Then carefully seal beam capsule inside two 000 gelatin bottoms, which have been filled with LR White. Make sure you seal well with LR White. Use regular mold holders to hold capsules. OR if you need to centrifuge material and you have a large sample size, leave cells in original 00-gelatin capsule (i.e. complete step 8). Then place 00 capsule inside a BEEM capsule that has been cut to accommodate 00 gelatin capsule (i.e. top half is removed); centrifuge inside a 12-15ml centrifuge tube {with a cotton bottom pad}, using a clinical centrifuge, until sample is spun down.

a. Use glass vials to mix.
b. mix solutions at RT
c. keep out of direct light
d. Use a Rotamix, as solution is not very miscible & needs to be GENTLY stirred.
e. KEEP Oxygen out of LRWhite &avoid excess air bubbles in resin. The dehydration and embedding are fairly straightforward.
f. TEST one block for sectioning quality. Polymerize another hour, if necessary.
g. LR White blocks should be stored at -20oC to preserve antigenicity indefinitely. Remove the gelatin and expose block to air in order to stop polymerization. (Newmann, p.104.)
h. FOR LIGHT STAIN, to see 5nm GOLD PARTICLES, use 2%UAaq for 10 min
i. FOR STAIN, to see 12nm GOLD PARTICLES, use 2%UAaq for 10 min and then 10” with Reymold’s LC.

References:
Griffiths, G., Fine Structure Immunocytochemistry, Springer-Verlag, 1993

Guthrie, C. and G.R. Fink, "Guide to Yeast Genetics and Molecular Biology, V194 in Methods in Enzymology, Academic press, 1991, Chapters 41&42.

Hyatt, A.D. "Immunogold Labeling Techniques", Chapter 4 in Electron Microscopy in Biology, Harris, J.R (ed.), IRL Press, 1991

Newman, G.R. and H.A. Hobot., Resin Microscopy and On-Section Immunocytochemistry, Springer-Verlag, 1993




IEM Protocol: section staining LR White sections, mounted on shiny side of Ni Grids. Whole cells pellet from Suti – arsenic affect on lysosomal bodies

1. If background labeling is a real problem, add a quenching step. Try quenching the thin sections with 0.1M Glycine in PBS/TBS before IEM. Sometimes, just the presence of BSA in the PBS or TBS buffer during section labeling is enough to block the free aldehyde groups. (Newman pp. 53 &135; Hyatt, p.62; Griffiths, pp.73-74). You will have to experiment with this step.

2. Place grid on drop of TBS or PBS for 5 min. Remove excess fluid: hold grid with forceps and bang fist on lab bench to shake off drop.

3. Blocking treatment: Place grid on drop of Blocking solution (PBS or TBS /1%BSA) for 5-10 min

4. Drain, but do not rinse grid and incubate in primary antibody(15-20ul drop) diluted 1:50 or 1:100 in PBS or TBS antibody buffer for 1-2hr hr at RT.

5. Rinse 2X (10"rinse and 2'soak each time).

6. Incubate in diluted Au-labelled secondary antibody at manufacture's recommended dilution (@1:25 to1:50 (sometimes as high as 1:10 to 1:25 for LRWhite sections) 1 hour at RT in the dark.

7. PBS or TBS rinses 2X (10"rinse or 2'soak each time).

8. H20 rinse and air dry.

9. .Section Stain: 2% aqueous Uranyl Acetate - 20mins-40 min. Reynold's Lead Citrate - 15-30" max or no LC
Notes
1. All wash steps and antibody incubations are done at room temp
2. Remove excess fluid: hold grid with forceps and bang fist on lab bench to shake off drop. Do this for each step.

Antibody Dilution and Washing Solutions:
Blocking solution(TBS type): 20mM Tris-HCL buffer(pH 7.5) with 0.9 % NaCl & 1%BSA & 0.02% NaAzide. (re-adjust pH after additions). You can substitute 1% BSA with 0.5-1% Fish gelatin(Recommended by griffiths, pp.252-254). You can
also use 2%(or a 1/20 dilution) normal serum(non-immune serum from the species that produced the secondary antibody) diluted in solution used for 1o antibody dilution, as a blocking solution.


Blocking solution(PBS type): 0.01M Phosphate buffer(pH 7.4) with 0.9 % NaCl & 1%BSA & 0.02% NaAzide. (Adjust pH after additions). You can substitute 1% BSA with 0.5-1% Fish gelatin(Recommended by griffiths, pp.252-254). You can
also use 2%(or a 1/20 dilution) normal serum(non-immune serum from the species that produced the secondary antibody) diluted in solution used for 1o antibody dilution, as a blocking solution.


TBS/BSA for primary antibody dilutions: 20mM Tris-HCL buffer(pH 7.5) with 0.9 % NaCl & 0.1%BSA(or 0.1% Fish gelatin) & 0.02% NaAzide. (re-adjust pH after additions)

PBS/BSA for primary antibody dilutions: 0.01M Phosphate buffer(pH 7.4) with 0.9% NaCl & 0.1%BSA(or 0.1% Fish gelatin) & 0.02% NaAzide. (Adjust pH after additions)

TB/BSA for secondary antibody dilutions: 20mM Tris-HCL buffer(pH 8.2) with 1%BSA & 0.02% NaAzide. (re-adjust pH after additions). Tris buffer without saline is preferred to highly ionic solutions as a diluent for colloidal gold, to avoid particle aggregation.

TB for washing: 20mM Tris-HCL buffer(pH 8.2).

References:
Griffiths, G., Fine Structure Immunocytochemistry, Springer-Verlag, 1993

Guthrie, C. and G.R. Fink, "Guide to Yeast Genetics and Molecular Biology, V194 in Methods in Enzymology, Academic press, 1991, Chapters 41&42.

Hyatt, A.D. "Immunogold Labeling Techniques", Chapter 4 in Electron Microscopy in Biology, Harris, J.R (ed.), IRL Press, 1991

Newman, G.R. and H.A. Hobot., Resin Microscopy and On-Section Immunocytochemistry, Springer-Verlag, 1993