Millipore Sigma Vibrant Logo
Attention: We have moved. Merck Millipore products are no longer available for purchase on MerckMillipore.com.Learn More

PC38 Anti-c-Fos (Ab-5) (4-17) Rabbit pAb

Anti-c-Fos (Ab-5) (4-17) Rabbit pAb MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
PC38
  
Purchase on Sigma-Aldrich

Overview

Replacement Information

Key Spec Table

Host
Rb
Description
Overview

This product has been discontinued.



We are offering Anti-c-Fos Antibody (Cat. No. ABE457) as a possible alternative. Please read the alternative product documentation carefully and contact technical service if you need additional information.






Recognizes the ~55 kDa c-Fos and ~62 kDa v-Fos proteins. Does not cross-react with the ~39 kDa Jun protein.
Catalogue NumberPC38
Brand Family Calbiochem®
Application Data
Detection of human c-Fos by staining free-floating sections. Sample: tissue sections from floating rat brain. Primary antibody: Anti-c-Fos (Ab-5) (4-17) Rabbit pAb (Cat. No. PC38) (1:20,000). Detection: DAB.
References
ReferencesBerghorn, K.A. et al. 1994. J. Histochem. Cytochem. 12, 1635.
Natoli, G., et al. 1993. Mol. Cell. Biol. 14, 989.
Trouche, D., et al. 1993. Nature 363, 79.
Wu, R.Y.-H., et al. 1993. Oncogene 8, 2237.
Colotta, F., et al. 1992. J. Biol. Chem. 267, 18278.
Hoffman, G.E., et al. 1992. in NEUROPROTOCOLS: A Companion to Methods in Neruosciences 1, 52.
Distel, R.J., and Spiegelman, B., 1990. Adv. Cancer Res. 55, 37.
Ransone, L.J., and Verma, I.M., 1990. Ann. Rev. Cell Biol. 6, 539.
DeTogni, P., et al. 1988. Mol. Cell. Biol. 8, 2251.
Greenberg, M., et al. 1984. Nature, London 311, 433.
Finkel, M.P., et al. 1966. Science 151, 698.
Product Information
FormLiquid
FormulationUndiluted serum.
Positive controlRat brain sections induced for Fos expression
PreservativeNone
Applications
Application ReferencesStaining Protocol 2 Le, W.W., et al. 1999. Endocrinology 140, 510. Rinaman, L., et al. 1997. Neuroscience 79, 1165. Graham, J.C., et al. 1995. J. Autonom. Nerv. System 55, 92. Rinaman, L., et al. 1995. J. Comp. Neurol. 360, 246. Berghorn, K.A., et al. 1994. Cytochem 42, 1635. Sved, A.F., et al. 1994. American Journal of Physiology. 266, R361. Lee, W.-S., et al. 1993. Endocrinology 133, 2248. Rinaman, L., et al. 1993. Journal of Comparative Neurology 338, 475. Lee, W.-S., et al. 1992. J. of Neuroendocrinology 4, 161. Olson, B.R., et al. 1992. Brain Research 569, 238. Pezzone, M.A., et al. 1992. Brain Research 597, 41. Hoffman, G.E., et al. 1991. Endocrinology 129, 3227. Verbalis, J.G., et al. 1991. J. Neuroendocrinology 3, 205. Free Floating Sections Dai, Y., et al. 2004. J. Neurosci. 24, 4293.
Key Applications Free-floating Sections
Application NotesFree-floating Sections (see comments and application references)
Application CommentsThis antibody has been screened for positive reactivity with floating rat brain sections induced for c-Fos expression. It will react with tissues treated with multiple fixatives including formalin, paraformaldehyde and acrolein. Does not react with the 39,000 c-Jun protein. Antibody should be titrated for optimal results in individual systems.

Protocol 1: Staining c-Fos Induced Formalin-fixed, Floating Rat Brain Sections with c-Fos (Ab-5)

MATERIALS NEEDED
Rats induced for c-Fos expression, 0.9% NaCl, Phosphate-buffered 4% paraformaldehyde, pH 7.0 (e.g. Accustain), 30% sucrose, PBS, pH 7.0, PBS/azide (PBS/0.02% NaN3), 0.3% H2O2, Blocking Solution (PBS/3% goat serum/0.25% Triton® X-100 detergent), PBS/0.1% NaN3/0.25% Triton® X-100 detergent (optional), PBS/0.5% Triton® X-100 detergent, Second step detection system (e.g. UniTect, Cat. No. XHC02), DAB substrate, DAB solution (0.04% DAB in PBS/0.02% H2O2), Ethanol: 50%, 75%, 95%, 100%, Xylene, Glass slides (subbed), Coverslips and Aqueous mounting medium (e.g. Permount)

EQUIPMENT NEEDED
Freezing microtome and microscope

Procedure

SAMPLE PREPARATION
1. Anesthetize rats, which have previously been prepared for c-Fos expression.
2. Perfuse animals transcardially with 0.9% saline for 10 min.
3. Perfuse animals with 500 ml phosphate-buffered 4% paraformaldehyde, pH 7.0 (10% neutral buffered formalin; e.g. Accustain).
4. Remove brains and cut into blocks with identifiable landmarks.
5. Submerge blocks in 30% sucrose overnight.
6. Cut a series of 4 coronal sections with a thickness of 40 µm each with a freezing microtome.
7. Store the sections in PBS/azide in tissue culture dishes at 4°C until used.

STAINING PROCEDURE
8. Rinse sections twice, 5 min each rinse, with PBS.
9. Incubate sections with 0.3% H2O2 in PBS for 30 min at room temperature.
10. Rinse sections twice, 5 min each rinse, with PBS.
11. Incubate sections for 2 h at room temperature in blocking solution.
12. Incubate sections with the Fos antibody diluted as suggested in blocking solution for 48 h at 4°C, or in PBS/0.1% azide/0.25% Triton® X-100 detergent overnight at room temp on a shaker. Note: It is strongly suggested that the primary antibody be titrated for optimal reactivity. Antibodies used at too high or too low concentration will often show reduced specific staining.
13. Rinse sections 6 × 10 min with PBS.
14. Prepare the detection reagent (horseradish peroxidase avidin-biotin complex) according to the manufacturers instructions.
15. Incubate the sections with the biotinylated goat anti-rabbit secondary antibody, diluted 1:600 in blocking solution, for 2 h at room temperature.
16. Rinse sections 3 × 10 min.
17. Incubate sections with the horseradish peroxidase avidin-biotin complex diluted in PBS/0.5% Triton® X-100 detergent for 1 h at room temperature.
18. Rinse sections 2 × 10 min in PBS.
19. Incubate sections in DAB solution for 6-10 min at room temperature.
20. Rinse sections twice, 5 min each rinse, with PBS.
21. Transfer the sections to subbed slides, allow to air dry.
22. Dehydrate slides in graded alcohols (50%, 75%, 95%, 100%) for 5 min each.
23. Incubate slides in xylene.
24. Mount coverslip slides with Permount and examine by microscopy.

Staining Protocol 2
Day One
For acrolein fixed tissue:

-thoroughly rinse out cryoprotectant 6 × 10 min washes in 0.05 M KPBS
-incubate 20 min in sodium borohydride (0.1 g in 10 ml KPBS)
-rinse multiple times until bubbles are gone; about 10 changes of KPBS; Proceed to primary antiserum step below:

(If blood is a problem, incubate 15 min in phenylhydrazine (0.014% in KPBS). Rinse multiple times.)

For tissue fixed with only paraformaldehyde:
-thoroughly rinse out cryoprotectant; 6 × 10 min washes in KPBS
(If blood is a problem, incubate 15 min in phenylhydrazine (0.014% in KPBS). Rinse multiple times.)

-Incubate in primary antibody in KPBS + 0.4% Triton® X-100 detergent; for 1 h at room temp, then 48 h at 4°C (72 h OK if start on Fri).
Day Two (48 h later)
-rinse 10 × 6 min (10 times over 60 min) in KPBS
-incubate in biotinylated-secondary anti-IgG, 1:600 in KPBS + 0.4% Triton® X-100 detergent for 1 h
-rinse 5 × 10 min in KPBS (prepare A/B solution at the beginning of the rinses; let stand > 30 min before use):
Start with 10 ml of KPBS + 0.4% Triton® X-100 detergent and add 45 µl of solution A, add 45 µl of solution B
-incubate in A/B (ELITE) solution for 1 h
-rinse 3 × 5 min in KPBS
-rinse 3 × 5 min in sodium acetate
-incubate in Ni sulfate-DAB chromogen solution about 10-15 min (time will vary)
-rinse 3 × 5 min in Sodium acetate to stop reaction
-rinse 3 × 5 min in KPBS
END OF FIRST STAIN
-mount, OR
-When double labeling: incubate in next primary antibody in KPBS + 0.4% Triton® X-100 detergent as above (1 h, RT then 48 h in cold)

Day Three (48 h later)
-rinse 10 × 6 min in KPBS
-incubate in biotin–anti-IgG 1:600 in KPBS + 0.4% Triton® X-100 detergent for 1 h
-rinse 5 × 10 min in KPBS (prepare A/B solution at the beginning of the rinses; let stand > 30 min
before use): Start with 10 ml of KPBS + 0.4% Triton® X-100 detergent and add 45 µl of solution A, add 45 µl of solution B
-incubate in A/B (Elite) solution for 1 h
-rinse 3 × 5 min in KPBS
-rinse 3 × 5 min in Tris (0.769 g Tris pH 7.2 per 100 ml saline)
-incubate in DAB chromogen solution 4-15 min (time may vary with antibody)
-rinse 3 × 5 min in Tris buffer to stop reaction
-rinse 3 × 5 min in KPBS
-mount from saline
-dehydrate, clear, and coverslip

Reagents:
0.05 M Potassium Phosphate Buffered Saline, KPBS (0.05 M)
9 g NaCl, 80 ml dibasic 0.5M KPO4 stock solution, 20 ml monobasic 0.5M KPO4 stock solution, add distilled H2O to a total volume of 1000 ml

Stock solutions:
Dibasic potassium phosphate stock (0.5M) g
87.09g Anhydrous K2HPO4 (Formula weight: 174.18) or 114.1g K2HPO4. 3 H2O (Formula weight: 228.23), 1000 ml distilled H2O

Monobasic potassium phosphate stock (0.5M)
34.02 g KH2PO4.H2O (Formula weight: 136.09), 500 ml distilled H2O

0.175 M Sodium Acetate in distilled water; (DO NOT USE saline or phosphate buffers in this solution!!! Do not buffer it, just use as is)

ABC Peroxidase 'Elite' kit ; Vector Laboratories, Burlingame, CA

Chromogen solutions
Nickel-DAB
250 mg Nickel (II) Sulfate, 4 mg DAB (Fluka tetrahydrochloride), 10 ml sodium acetate, 8.3 µl 30% H2O2
DAB
4 mg DAB, 10 ml Tris buffer (pH 7.2)–must be at room temperature not refrigerated!!! 8.3 µl 30% H2O2

Antifreeze Cryoprotectant: Antifreeze to prevent freezing for tissue section storage:
500 ml 0.1 M sodium phosphate buffer pH 7.2, (1.59 g NaH2PO4
5.47 g Na2HPO4), 500 ml dH20, 300 g sucrose, 10 g polyvinylpyrrolidone (PVP-40) (optional), 300 ml ethylene glycol

Adjust volume to 1000 ml with distilled water

Recommended Fixative:
Best results are obtained if animals are perfused rather than immersion fixed. Best fix: 2.5% Acrolein, (Polysciences, EM grade) in 4% Paraformaldehyde, pH 6.8; also acceptable: 4% Paraformaldehyde, pH 6.8. (higher pH can be source of non-specific background)
Fixation with Acrolein containing fixatives must be done in a hood (approved for proper air flow). Solutions of acrolein are neutralized with 10% sodium bisulfite. Vats of the latter solution are always on hand during a perfusion.
(Use of fixatives without acrolein may increase background staining and reduce fos antigenicity.)

Sectioning: Sections should be submerged in aqueous sucrose (30%) and cut on a sliding microtome (freezing microtome) or cryostat set at 25 µm. The sections are placed immediately into the cryoprotectant/antifreeze solution. Sections can be stored indefinitely in cryoprotectant. If a vibratome is used sections should ideally be no more than 30 µm in thickness. Thicker sections are possible but all rinses must be lengthened. Sections mounted on slides (eg normal cryostat sections) will require higher concentrations of antibodies.

A note on mounting and coverslipping: After sections are mounted onto subbed glass slides, they are airdried overnight. They then are placed sequentially for about 5-10 min each in distilled water, 50% ethanol, 70% and 95%. Then they are placed into 2 changes of bottled absolute alcohol with about 10-15 min each (the stuff that usually comes in plastic jugs has too much water and is more like 95% that 100%). Next clear the sections in either xylenes or histoclear (if the solution turns cloudy and doesn't clear immediately, there is too much water in the 100% solutions, change them and start the last dehydration steps over. If the tissue isn't very clear when placed in the solution, then go backwards to 70% alcohol and go much slower at the higher alcohol solutions. Use mounting medium of choice to set coverslip; allow slide to dry prior to analysis.

A note on titrating antibodies
Always run the following series using ABC and NiDAB
1:1000, 1:3000; 1:10,000, 1:30,000; 1:100,000; 1:300,000.
Further dilutions may be possuble but should be determined by end-user. Important: staining time is held constant at 15 min for titrations.

Note on buffers: KPBS is used due to the fact that stock solutions of mono and dibasic potassium salts do not percipitate upon storage.
Biological Information
Immunogena synthetic peptide (SGFNADYEASSSRC) (Cat. No. PP10) corresponding to amino acids 4-17 of human c-Fos
ImmunogenHuman
HostRabbit
IsotypeIgG
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Storage and Shipping Information
Ship Code Dry Ice Only
Toxicity Standard Handling
Storage -20°C
Avoid freeze/thaw Avoid freeze/thaw
Do not freeze Ok to freeze
Special InstructionsFollowing intitial thaw, aliquot and freeze (-20°C).
Packaging Information
Transport Information
Supplemental Information
Specifications
Global Trade Item Number
Catalogue Number GTIN
PC38 0

Documentation

Anti-c-Fos (Ab-5) (4-17) Rabbit pAb Certificates of Analysis

TitleLot Number
PC38

References

Reference overview
Berghorn, K.A. et al. 1994. J. Histochem. Cytochem. 12, 1635.
Natoli, G., et al. 1993. Mol. Cell. Biol. 14, 989.
Trouche, D., et al. 1993. Nature 363, 79.
Wu, R.Y.-H., et al. 1993. Oncogene 8, 2237.
Colotta, F., et al. 1992. J. Biol. Chem. 267, 18278.
Hoffman, G.E., et al. 1992. in NEUROPROTOCOLS: A Companion to Methods in Neruosciences 1, 52.
Distel, R.J., and Spiegelman, B., 1990. Adv. Cancer Res. 55, 37.
Ransone, L.J., and Verma, I.M., 1990. Ann. Rev. Cell Biol. 6, 539.
DeTogni, P., et al. 1988. Mol. Cell. Biol. 8, 2251.
Greenberg, M., et al. 1984. Nature, London 311, 433.
Finkel, M.P., et al. 1966. Science 151, 698.

Brochure

Title
Caspases and other Apoptosis Related Tools Brochure

Citations

Title
  • Yi Dai, et al. (2004) Proteinase-activated receptor 2-mediated potentiation of transient receptor potential vanilloid subfamily 1 activity reveals a mechanism for proteinase-induced inflammatory pain. Journal of Neuroscience 24, 4293-4299.
    		•
    Data Sheet

    Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

    Revision30-September-2008 JSW
    ApplicationFree-floating Sections (see comments and application references)
    Application Data
    Detection of human c-Fos by staining free-floating sections. Sample: tissue sections from floating rat brain. Primary antibody: Anti-c-Fos (Ab-5) (4-17) Rabbit pAb (Cat. No. PC38) (1:20,000). Detection: DAB.
    DescriptionRabbit polyclonal supplied as undiluted serum. Recognizes the ~55 kDa c-Fos and ~62 kDa v-Fos proteins.
    BackgroundThe c-fos proto-oncogene and transcription factor has been implicated in cell growth, differentiation and development. fos, an immediate early gene, is induced by a large number of stimuli ranging from mitogens, differentiation-specific agents, pharmacological compounds and other factors. Induction of the 62,000 dalton c-Fos protein is rapid but transient. The c-Fos protein associates with the 39,000 dalton c-Jun protein which has been identified as a proto-oncogene and transcription factor. The Fos/Jun protein complex binds specifically to a sequence element in DNA referred to as the AP-1 binding site.
    HostRabbit
    Immunogen speciesHuman
    Immunogena synthetic peptide (SGFNADYEASSSRC) (Cat. No. PP10) corresponding to amino acids 4-17 of human c-Fos
    IsotypeIgG
    Specieshuman, mouse, rat
    Positive controlRat brain sections induced for Fos expression
    FormLiquid
    FormulationUndiluted serum.
    PreservativeNone
    CommentsThis antibody has been screened for positive reactivity with floating rat brain sections induced for c-Fos expression. It will react with tissues treated with multiple fixatives including formalin, paraformaldehyde and acrolein. Does not react with the 39,000 c-Jun protein. Antibody should be titrated for optimal results in individual systems.

    Protocol 1: Staining c-Fos Induced Formalin-fixed, Floating Rat Brain Sections with c-Fos (Ab-5)

    MATERIALS NEEDED
    Rats induced for c-Fos expression, 0.9% NaCl, Phosphate-buffered 4% paraformaldehyde, pH 7.0 (e.g. Accustain), 30% sucrose, PBS, pH 7.0, PBS/azide (PBS/0.02% NaN3), 0.3% H2O2, Blocking Solution (PBS/3% goat serum/0.25% Triton® X-100 detergent), PBS/0.1% NaN3/0.25% Triton® X-100 detergent (optional), PBS/0.5% Triton® X-100 detergent, Second step detection system (e.g. UniTect, Cat. No. XHC02), DAB substrate, DAB solution (0.04% DAB in PBS/0.02% H2O2), Ethanol: 50%, 75%, 95%, 100%, Xylene, Glass slides (subbed), Coverslips and Aqueous mounting medium (e.g. Permount)

    EQUIPMENT NEEDED
    Freezing microtome and microscope

    Procedure

    SAMPLE PREPARATION
    1. Anesthetize rats, which have previously been prepared for c-Fos expression.
    2. Perfuse animals transcardially with 0.9% saline for 10 min.
    3. Perfuse animals with 500 ml phosphate-buffered 4% paraformaldehyde, pH 7.0 (10% neutral buffered formalin; e.g. Accustain).
    4. Remove brains and cut into blocks with identifiable landmarks.
    5. Submerge blocks in 30% sucrose overnight.
    6. Cut a series of 4 coronal sections with a thickness of 40 µm each with a freezing microtome.
    7. Store the sections in PBS/azide in tissue culture dishes at 4°C until used.

    STAINING PROCEDURE
    8. Rinse sections twice, 5 min each rinse, with PBS.
    9. Incubate sections with 0.3% H2O2 in PBS for 30 min at room temperature.
    10. Rinse sections twice, 5 min each rinse, with PBS.
    11. Incubate sections for 2 h at room temperature in blocking solution.
    12. Incubate sections with the Fos antibody diluted as suggested in blocking solution for 48 h at 4°C, or in PBS/0.1% azide/0.25% Triton® X-100 detergent overnight at room temp on a shaker. Note: It is strongly suggested that the primary antibody be titrated for optimal reactivity. Antibodies used at too high or too low concentration will often show reduced specific staining.
    13. Rinse sections 6 × 10 min with PBS.
    14. Prepare the detection reagent (horseradish peroxidase avidin-biotin complex) according to the manufacturers instructions.
    15. Incubate the sections with the biotinylated goat anti-rabbit secondary antibody, diluted 1:600 in blocking solution, for 2 h at room temperature.
    16. Rinse sections 3 × 10 min.
    17. Incubate sections with the horseradish peroxidase avidin-biotin complex diluted in PBS/0.5% Triton® X-100 detergent for 1 h at room temperature.
    18. Rinse sections 2 × 10 min in PBS.
    19. Incubate sections in DAB solution for 6-10 min at room temperature.
    20. Rinse sections twice, 5 min each rinse, with PBS.
    21. Transfer the sections to subbed slides, allow to air dry.
    22. Dehydrate slides in graded alcohols (50%, 75%, 95%, 100%) for 5 min each.
    23. Incubate slides in xylene.
    24. Mount coverslip slides with Permount and examine by microscopy.

    Staining Protocol 2
    Day One
    For acrolein fixed tissue:

    -thoroughly rinse out cryoprotectant 6 × 10 min washes in 0.05 M KPBS
    -incubate 20 min in sodium borohydride (0.1 g in 10 ml KPBS)
    -rinse multiple times until bubbles are gone; about 10 changes of KPBS; Proceed to primary antiserum step below:

    (If blood is a problem, incubate 15 min in phenylhydrazine (0.014% in KPBS). Rinse multiple times.)

    For tissue fixed with only paraformaldehyde:
    -thoroughly rinse out cryoprotectant; 6 × 10 min washes in KPBS
    (If blood is a problem, incubate 15 min in phenylhydrazine (0.014% in KPBS). Rinse multiple times.)

    -Incubate in primary antibody in KPBS + 0.4% Triton® X-100 detergent; for 1 h at room temp, then 48 h at 4°C (72 h OK if start on Fri).
    Day Two (48 h later)
    -rinse 10 × 6 min (10 times over 60 min) in KPBS
    -incubate in biotinylated-secondary anti-IgG, 1:600 in KPBS + 0.4% Triton® X-100 detergent for 1 h
    -rinse 5 × 10 min in KPBS (prepare A/B solution at the beginning of the rinses; let stand > 30 min before use):
    Start with 10 ml of KPBS + 0.4% Triton® X-100 detergent and add 45 µl of solution A, add 45 µl of solution B
    -incubate in A/B (ELITE) solution for 1 h
    -rinse 3 × 5 min in KPBS
    -rinse 3 × 5 min in sodium acetate
    -incubate in Ni sulfate-DAB chromogen solution about 10-15 min (time will vary)
    -rinse 3 × 5 min in Sodium acetate to stop reaction
    -rinse 3 × 5 min in KPBS
    END OF FIRST STAIN
    -mount, OR
    -When double labeling: incubate in next primary antibody in KPBS + 0.4% Triton® X-100 detergent as above (1 h, RT then 48 h in cold)

    Day Three (48 h later)
    -rinse 10 × 6 min in KPBS
    -incubate in biotin–anti-IgG 1:600 in KPBS + 0.4% Triton® X-100 detergent for 1 h
    -rinse 5 × 10 min in KPBS (prepare A/B solution at the beginning of the rinses; let stand > 30 min
    before use): Start with 10 ml of KPBS + 0.4% Triton® X-100 detergent and add 45 µl of solution A, add 45 µl of solution B
    -incubate in A/B (Elite) solution for 1 h
    -rinse 3 × 5 min in KPBS
    -rinse 3 × 5 min in Tris (0.769 g Tris pH 7.2 per 100 ml saline)
    -incubate in DAB chromogen solution 4-15 min (time may vary with antibody)
    -rinse 3 × 5 min in Tris buffer to stop reaction
    -rinse 3 × 5 min in KPBS
    -mount from saline
    -dehydrate, clear, and coverslip

    Reagents:
    0.05 M Potassium Phosphate Buffered Saline, KPBS (0.05 M)
    9 g NaCl, 80 ml dibasic 0.5M KPO4 stock solution, 20 ml monobasic 0.5M KPO4 stock solution, add distilled H2O to a total volume of 1000 ml

    Stock solutions:
    Dibasic potassium phosphate stock (0.5M) g
    87.09g Anhydrous K2HPO4 (Formula weight: 174.18) or 114.1g K2HPO4. 3 H2O (Formula weight: 228.23), 1000 ml distilled H2O

    Monobasic potassium phosphate stock (0.5M)
    34.02 g KH2PO4.H2O (Formula weight: 136.09), 500 ml distilled H2O

    0.175 M Sodium Acetate in distilled water; (DO NOT USE saline or phosphate buffers in this solution!!! Do not buffer it, just use as is)

    ABC Peroxidase 'Elite' kit ; Vector Laboratories, Burlingame, CA

    Chromogen solutions
    Nickel-DAB
    250 mg Nickel (II) Sulfate, 4 mg DAB (Fluka tetrahydrochloride), 10 ml sodium acetate, 8.3 µl 30% H2O2
    DAB
    4 mg DAB, 10 ml Tris buffer (pH 7.2)–must be at room temperature not refrigerated!!! 8.3 µl 30% H2O2

    Antifreeze Cryoprotectant: Antifreeze to prevent freezing for tissue section storage:
    500 ml 0.1 M sodium phosphate buffer pH 7.2, (1.59 g NaH2PO4
    5.47 g Na2HPO4), 500 ml dH20, 300 g sucrose, 10 g polyvinylpyrrolidone (PVP-40) (optional), 300 ml ethylene glycol

    Adjust volume to 1000 ml with distilled water

    Recommended Fixative:
    Best results are obtained if animals are perfused rather than immersion fixed. Best fix: 2.5% Acrolein, (Polysciences, EM grade) in 4% Paraformaldehyde, pH 6.8; also acceptable: 4% Paraformaldehyde, pH 6.8. (higher pH can be source of non-specific background)
    Fixation with Acrolein containing fixatives must be done in a hood (approved for proper air flow). Solutions of acrolein are neutralized with 10% sodium bisulfite. Vats of the latter solution are always on hand during a perfusion.
    (Use of fixatives without acrolein may increase background staining and reduce fos antigenicity.)

    Sectioning: Sections should be submerged in aqueous sucrose (30%) and cut on a sliding microtome (freezing microtome) or cryostat set at 25 µm. The sections are placed immediately into the cryoprotectant/antifreeze solution. Sections can be stored indefinitely in cryoprotectant. If a vibratome is used sections should ideally be no more than 30 µm in thickness. Thicker sections are possible but all rinses must be lengthened. Sections mounted on slides (eg normal cryostat sections) will require higher concentrations of antibodies.

    A note on mounting and coverslipping: After sections are mounted onto subbed glass slides, they are airdried overnight. They then are placed sequentially for about 5-10 min each in distilled water, 50% ethanol, 70% and 95%. Then they are placed into 2 changes of bottled absolute alcohol with about 10-15 min each (the stuff that usually comes in plastic jugs has too much water and is more like 95% that 100%). Next clear the sections in either xylenes or histoclear (if the solution turns cloudy and doesn't clear immediately, there is too much water in the 100% solutions, change them and start the last dehydration steps over. If the tissue isn't very clear when placed in the solution, then go backwards to 70% alcohol and go much slower at the higher alcohol solutions. Use mounting medium of choice to set coverslip; allow slide to dry prior to analysis.

    A note on titrating antibodies
    Always run the following series using ABC and NiDAB
    1:1000, 1:3000; 1:10,000, 1:30,000; 1:100,000; 1:300,000.
    Further dilutions may be possuble but should be determined by end-user. Important: staining time is held constant at 15 min for titrations.

    Note on buffers: KPBS is used due to the fact that stock solutions of mono and dibasic potassium salts do not percipitate upon storage.
    Storage Avoid freeze/thaw
    -20°C
    Do Not Freeze Ok to freeze
    Special InstructionsFollowing intitial thaw, aliquot and freeze (-20°C).
    Toxicity Standard Handling
    ReferencesBerghorn, K.A. et al. 1994. J. Histochem. Cytochem. 12, 1635.
    Natoli, G., et al. 1993. Mol. Cell. Biol. 14, 989.
    Trouche, D., et al. 1993. Nature 363, 79.
    Wu, R.Y.-H., et al. 1993. Oncogene 8, 2237.
    Colotta, F., et al. 1992. J. Biol. Chem. 267, 18278.
    Hoffman, G.E., et al. 1992. in NEUROPROTOCOLS: A Companion to Methods in Neruosciences 1, 52.
    Distel, R.J., and Spiegelman, B., 1990. Adv. Cancer Res. 55, 37.
    Ransone, L.J., and Verma, I.M., 1990. Ann. Rev. Cell Biol. 6, 539.
    DeTogni, P., et al. 1988. Mol. Cell. Biol. 8, 2251.
    Greenberg, M., et al. 1984. Nature, London 311, 433.
    Finkel, M.P., et al. 1966. Science 151, 698.
    Citation
  • Yi Dai, et al. (2004) Proteinase-activated receptor 2-mediated potentiation of transient receptor potential vanilloid subfamily 1 activity reveals a mechanism for proteinase-induced inflammatory pain. Journal of Neuroscience 24, 4293-4299.
    		•
    Application referencesStaining Protocol 2 Le, W.W., et al. 1999. Endocrinology 140, 510. Rinaman, L., et al. 1997. Neuroscience 79, 1165. Graham, J.C., et al. 1995. J. Autonom. Nerv. System 55, 92. Rinaman, L., et al. 1995. J. Comp. Neurol. 360, 246. Berghorn, K.A., et al. 1994. Cytochem 42, 1635. Sved, A.F., et al. 1994. American Journal of Physiology. 266, R361. Lee, W.-S., et al. 1993. Endocrinology 133, 2248. Rinaman, L., et al. 1993. Journal of Comparative Neurology 338, 475. Lee, W.-S., et al. 1992. J. of Neuroendocrinology 4, 161. Olson, B.R., et al. 1992. Brain Research 569, 238. Pezzone, M.A., et al. 1992. Brain Research 597, 41. Hoffman, G.E., et al. 1991. Endocrinology 129, 3227. Verbalis, J.G., et al. 1991. J. Neuroendocrinology 3, 205. Free Floating Sections Dai, Y., et al. 2004. J. Neurosci. 24, 4293.