(Raleigh, JA, Chou, S-C, Tables, L, Suchindran, S and Varia, MA. Relationship of hypoxia to metallothionein expression in murine tumors. Int. J. Radiat. Oncol. Biol. Phys. 42: 727-730, 1998)
Note that the F(ab’)2 secondary reagent produces very clean images with extremely low background in mouse and human tissues. However, secondary strategies other than the F(ab’)2 approach can be used. In particular, the DAKO Catalyzed Signal Amplification System for mouse antibodies (Kit #K1500) is used routinely in our laboratories for clinical samples.
| Step | Procedure | Time,min. | Temp. | Reagents | Notes |
|---|---|---|---|---|---|
| 1 | Warm paraffinized tissue section | 20 | 40oC | None | |
| 2 | Dip and blot 10 times | 2 | RT | Clear-Rite 3 | 1 |
| 3 | " | 2 | RT | 100% Ethanol | |
| 4 | " | 2 | RT | 95% Aqueous ethanol | |
| 5 | " | 2 | RT | 80% Aqueous ethanol | |
| 6 | " | 2 | RT | Deionized water + 0.2% Brij 35 | 2 |
| 7 | " | 2 | RT | PBS+ 0.2% Brij 35 | 3 |
| 8 | Quench tissue peroxidase | 5 | RT | 3% H2O2 in distilled water | 4 |
| 9 | Antigen retrieval | 20 | 95o | Citrate Buffer pH6 | 5,6 |
| 10 | Wash | 2 | 0o | PBS + 0.2% Brij 35 | 7 |
| 11 | Block non-specific binding | 5 | RT | DAKO blocking solution | 8,9 |
| 12 | 1o MAb, slides horizontal | 40 | RT | Hypoxyprobe MAb1 (1/50) | 10 |
| 13 | Wash | 2 | 0o | PBS + 0.2% Brij 35 | 7 |
| 14 | 2o antibody | 10 | RT | Biotin-conjugated F(ab')2 (1/500) | 11 |
| 15 | Wash | 2 | 0o | PBS + 0.2% Brij 35 | 7 |
| 16 | Peroxidase | 10 | RT | Streptavidin peroxidase | 12 |
| 17 | Wash | 2 | 0o | PBS + 0.2% Brij 35 | 7 |
| 18 | Peroxidase chromogen | 10 | RT | DAB | 13 |
| 19 | Wash | 2 | RT | Distilled water | 14 |
| 20 | Counterstaining | 0.5 | RT | Aqua Hematoxylin | 15 |
| 21 | Wash | 2 | RT | Distilled water | 14 |
| 22 | Mount & dry slides | 20 | RT | Crystal/Mount | 16 |
| 1. | For deparaffinization, hydration and peroxidase quenching, steps 2-8, slides are held vertically. ProbeOn plus slides (cat# 15-188-52) and a MicroProbe Staining Station, both from Fisher Scientific, are suitable for these steps. Clear-Rite 3 is a non-toxic alternative to xylene and is available from Richard-Allan Scientific (Kalamazoo, MI)(cat.# 6901). back to steps |
| 2. | Brij 35 is enzyme grade polyoxyethylene(23)lauryl ether available from Fisher Scientific (cat.# BP345-500). back to steps |
| 3. | PBS is phosphate buffered saline, 10 mM, prepared from tablets available from Sigma (cat.# P-4417). back to steps |
| 4. | 3% H2O2 is diluted Analytical Reagent 31.3% H2O2 available from Malinckrodt Baker (Paris, KT)(cat.# 5240). back to steps |
| 5. | For example, AbD Serotech or DAKO Corp. back to steps |
| 6. | Slides held vertically in slide incubator. back to steps |
| 7. | Slides washed with magnetically stirred PBS + 0.2% Brij 35 in a Coplin jar held on ice. back to steps |
| 8. | Serum free protein blocker from DAKO Corp. (Carpinteria, CA)(cat. #X0909). back to steps |
| 9. | Slides held horizontally for steps 11-18 so as to limit non-specific, edge staining of the sections. back to steps |
| 10. | Exhausted supernatant containing 1oMAb diluted 1/50 in 10 mM PBS containing 0.2% Brij 35 and 1 drop of DAKO protein blocker/mL. Typically, 150 uL of diluted 1o MAb solution is applied to each tissue section. back to steps |
| 11. | A biotin-conjugated F(ab’)2 fragment of rabbit anti-mouse IgG is available from a number of suppliers including ABD Serotec (Raleigh, NC; www.ab-direct.com). Typically, the biotin conjugated F(ab’)2 fragment is diluted 1/500 in 10 mM PBS containing 0.2% Brij 35 and 1 drop of DAKO protein blocker/mL. When combined with peroxidase conjugated streptavidin, the biotin conjugated F(ab’)2 reagent produces very clean images with extremely low background signals. Secondary strategies other than the biotin conjugated F(ab')2 approach can also be used. In particular, DAKO Catalyzed Signal Amplification System for mouse antibodies (Kit #K1500) is used routinely in our laboratories for clinical samples. back to steps |
| 12. | Peroxidase conjugated streptavidin from DAKO (cat.#K1016). back to steps |
| 13. | Liquid 3,3'-diaminobenzidine reagent (DAB) from DAKO (cat.#K3465). back to steps |
| 14. | Slides washed with magnetically stirred distilled water at RT in a Coplin jar. back to steps |
| 15. | Aqua Hematoxylin from Innovex Science (Richmond, CA)(cat.#NB305). back to steps |
| 16. | Biomeda Crystal/Mount from Fisher Scientific (cat.#BM-M03). back to steps |
(A.S.E .Ljungdkvist et al. Changes in tumor hypoxia measured with a double hypoxic marker technique, Int. J. Radiat. Oncol. Biol. Phys. 48: 1529-1538, 2000).
Frozen sections are fixed in acetone for 10 minutes and washed in phosphate buffered saline, pH 7.4 (PBS).The sections are then incubated overnight at 4oC with mouse IgG1 anti-pimonidazole monoclonal antibody (NPI, Inc; diluted 1/10 in PBS).The sections are washed with PBS and incubated for 90 minutes at room temperature with Cy-3 conjugated goat antimouse antisera (Jackson Immuno Research Laboratories; diluted 1/150 in PBS).