Staining Protocol (normal ICC) (1,2)

Day One

For acrolein fixed tissue :

  • thoroughly rinse out cryoprotectant 6 x 10 min washes in .05 M KPBS
  • incubate 20 min in sodium borohydride (0.1g 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 x 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; for 1 hour at room temp, then 48 hours at 4 C (72 hrs OK if start on Fri).

Day Two (48 hr later)

  • rinse 10 x 6 min (10 times over 60 min) in KPBS
  • incubate in biotinylated-secondary anti- IgG, 1:600 in KPBS + 0.4% Triton-X for 1 hr
  • rinse 5 x 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

add 45 ul of solution A

add 45 ul of solution B

  • incubate in A/B (ELITE) solution for 1 hr
  • rinse 3 x 5 min in KPBS
  • rinse 3 x 5 min in sodium acetate
  • incubate in Ni sulfate-DAB chromogen solution about 10 –15 minutes (time will vary)
  • rinse 3 x 5 min in Sodium acetate to stop reaction
  • rinse 3 x 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 (10x)

20 ml monobasic 0.5M KPO4 stock solution (10x)

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

If a stock solution of DAB is desired, a 2% w/v solution of DAB is made in distilled water, aliquoted for easy use (0.1 ml will be added to 10 ml of desired solution) and then store at -20˚C.

Chromogen solutions

Nickel-DAB

250 mg Nickel (II) Sulfate (NOT nickel ammonium sulfate!)

2 mg DAB (Fluka tetrahydrochloride from Sigma)

10 ml sodium acetate

8.3 µl 3% H2O2

Antifreeze Cryoprotectant: Antifreeze to prevent freezing for tissue section storage:

500 ml 0.1M sodium phosphate buffer pH 7.2

1.59 g NaH2PO4

5.47 g Na2HPO4

500 ml dH2O

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 antigenicity; 2x more primary antibody may be needed to achieve the same staining level, but this is dependent upon the antigen being examined.)

Sectioning: Sections should be sunk in aqueous sucrose (30%) and cut on a sliding microtome (freezing microtome) or cryostat at 25 um. 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 um thick. Thicker is possible, but all rinses must be lengthened. Sections mounted on slides (e.g., normal cryostat sections) will require higher concentrations of antibodies (can be 10-20x).

1. Berghorn KA, Bonnet JH, Hoffman GE: cFos immunoreactivity is enhanced with biotin amplification. J. Histochem. Cytochem 1994, 42: 1635-1642.

2. Hoffman GE, Smith MS, Fitzsimmons MD: Detecting steroidal effects on immediate early gene expression in the hypothalamus. Neuroprotocols 1992, 1: 52-66.

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 minutes each (the stuff that usually comes in plastic jugs has too much water and is closer to 95% than 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 Permount or standard mounting medium to place coverglass, let dry and look at the pretty slides…

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

You can start with a more dilute concentration if someone recommended it (a good antibody will almost never titer at higher concentrations in my experience but will almost always fall at a much more dilute concentration than others recommend or have used). Important: staining time is held constant at 15 minutes for titrations.

Note on buffers: the reason we use KPBS is due to the fact that many, many years ago we made stock solutions of mono and dibasic salts. Sodium salts when placed in the refrigerator crystallized; potassium salts did not. We no longer make the stock solutions but have not changed the buffer since “if it ain’t broke, don’t fix it”. (it probably doesn’t matter though).

Note on use of cultured cells. For cultured tissue, one can fix with 4% paraformaldehyde for a couple of hrs (room temperature), or PF+acrolein for 5 minutes. Rinse the tissue multiple times and then cells can be placed into antifreeze and stored -20˚C until staining is desired. No prior placement in sucrose only is needed. To stain, if acrolein is used in the fixative, tissue should be rinsed free of antifreeze and treated with sodium borohydrate before application of the primary antibody. Like free floating sections, a titration of the antibody must be conducted as described above, and all other steps followed. It is easier to use cultures on coverglass so that xylene-based mounting media can be used for clearing. If on plastic, then use DAB not NiDAB, and mount in water-soluble media (niDAB will fade). Fluorescence is not recommended as the first pass for titer determination as it can be more difficult to establish a reliable concentration of primary antibody. For conversion to direct tagged florescence of secondary antibodies, the optimum concentration of primary is increased 10-20 fold. Incubation in primary antibody should be 24 hr (room temperature)-or 48 hr (4˚C); shorter times may not enable as good signal. Longer incubation times permit much lower concentrations of primary antibody as well. Converting data from peroxidase chromagen based staining to other fluorescence methods is outlined in the Current Protocols paper.

Ref’s

Verbalis, J.G., E.M. Stricker, A.G. Robinson, and G.E. Hoffman. Cholecystokinin activates cFos expression in hypothalamic oxytocin and corticotropin releasing hormone neurons. J. Neuroendocrinology 3: 205-213, 1991.

Hoffman, G.E., T.J. McDonald, R. Shedwick, and P.W. Nathanielsz. Activation of cFos in the ovine paraventricular CRH neurons at the time of parturition. Endocrinology 129: 3227-3233, 1991.

Lee, W.-S., M.S. Smith, and G.E. Hoffman. cFos activity identifies recruitment of LHRH neurons during the ascending phase of the proestrous luteinizing hormone surge. Journal of Neuroendocrinology 4: 161-166, 1992.

Olson, B.R., G.E. Hoffman, A.F. Sved, E.M. Stricker, and J.G. Verbalis. Cholecystokinin induces c-Fos expression in hypothalamic oxytocinergic neurons projecting to the dorsal vagal complex. Brain Research 569: 238-248, 1992

Pezzone, M.A., W.S. Lee, G.E. Hoffman, B.S. Rabin, Induction of c-Fos immunoreactivity in the rat forebrain by conditioned and unconditioned stimuli. Brain Research 597: 41-50, 1992.

Lee, W.-S., R. Abbud, G.E. Hoffman, and M.S. Smith, Effects of n-methyl-d-aspartate (NMDA) receptor activation on cFos expression in luteinizing hormone-releasing hormone neurons in female rats. Endocrinology 133: 2248-2254, 1993.

Roberts, M.M., A.G. Robinson, M.D. Fitzsimmons, W-S. Lee, and G.E.Hoffman. cFos expression in vasopressin and oxytocin magnocellular neurons reveals functional heterogeneity within magnocellular neurons. Neuroendocrinology 57: 388-400, 1993.

Rinaman, L., J.G.Verbalis, E.M. Stricker, and G.E. Hoffman, Distribution and neurochemical phenotypes of caudal medullary neurons activated to express cFos following perpheral administration of cholecystokinin. Journal of Comparative Neurology 338: 475-490, 1993.

Berghorn, K.A., J.H Bonnett, and G.E. Hoffman. cFos immunoreactivity is enhanced with biotin amplification. J. Histochem. Cytochem: 42:1635-1642, 1994.

Sved, A.F., D.L. Mancini, J.C. Graham, A.M. Schreihofer, and G.E. Hoffman. PNMT-containing neurons of the C1 cell group express c-Fos in response to changes in baroreceptor input. American Journal of Physiology.266: R361-367, 1994.

Rinaman, L., Hoffman, G.E., Dohanics, J., Le, W-W, Stricker, E.M., and Verbalis, J.G. Cholecystokinin activates catecholaminergic neurons in the caudal medulla that innervate the paraventricular nucleus of the hypothalamus in rats. J. Comp. Neurol. 360: 246-256, 1995

Rinaman, L., Stricker, E.M., Hoffman, G.E., Verbalis, J.G. Central cFos expression in neonatal and adult rats after central hypertonic saline. Neuroscience 79:1165-1175, 1997.

Graham, J.C., Hoffman, G.E., and Sved, A.F. cFos expression in brain in response to hypotension and hypertension in conscious rats. J. Autonom. Nerv. System. 55: 92-104, 1995.

Le, W.W., Berghorn, K.A., Rassnick, S., Dohanics, J., and Hoffman, G.E., Periventricular preoptic area neurons co-activated with LHRH neurons at the time of the LH surge are LHRH afferents. Endocrinology 140: 510-19, 1999

Hoffman, G.E., M.S. Smith, and M.D. Fitzsimmons. Detecting steroidal effects on immediate early gene expression in the hypothalamus. In, Neuroprotocols: A companion to Methods in Neuroscience. Volume 1 No. 2, Steroid Feedback Mechanisms, S.P. Kalra, Ed., 1992, Academic Press, New York, pp 52-66.

Hoffman, G.E., Sita L.V. and Le, WW. Importance of titrating antibodies. Current Protocols in Neuroscience Chapter 2 Unit 2.12, 2008. PMID: 18972376