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

Anti-c-Fos (Ab-5) (4-17) Rabbit pAb: SDB (Sicherheitsdatenblätter), Analysenzertifikate und Qualitätszertifikate, Dossiers, Broschüren und andere verfügbare Dokumente.
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      Übersicht

      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
      Bestellnummer GTIN
      PC38 0

      Documentation

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

      TitelChargennummer
      PC38

      Literatur

      Übersicht
      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.

      Broschüre

      Titel
      Caspases and other Apoptosis Related Tools Brochure

      Literaturstellen

      Titel
    • 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.
      		•
      Datenblatt

      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.
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