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For induction of c-fos protein activity rats were injected with 1.0 ml of 1.5 M NaCl per 100 grams of body weight. Negative control rats were injected with the same volume of normal saline. The ImmunoStar c-fos antiserum was quality control tested using standard immunohistochemical methods. The antiserum demonstrates strongly positive labeling of rat paraventricular nucleus and supraoptic nucleus using indirect immunofluorescent and biotin/avidin-HRP techniques. No labeling was seen in negative control rats. Recommended primary dilutions for these methods are 1/4000-1/6000 in PBS/0.3% Triton X-100 - FITC Technique and 1/4000-1/6000 in PBS/0.3% Triton X-100 - biotin/avidin-HRP Technique.
Injection of intraperitoneal hypertonic saline to provoke fos expression is most usually used to set ICC staining parameters. It is critical that tissue is harvested 60-90 minutes post injection for this and any other stress that is used. Inject 1 ml/100 g body weight 1.5 M NaCl intraperitoneally. Harvest brains 90 minutes post injection. Time is critical.
Specificity of the antiserum was demonstrated by blockage of staining in experimental rats by omission of c-fos antibody or by substitution of antibody pre-incubated with synthetic peptide or the conjugate. Immunoblot analysis of mediobasal hypothalamus showed a single band of approximately 55-60 kD.
Photo Description: IHC image of neurons staining for c-Fos in the rat supraoptic nucleus. The tissue was fixed with 4% formaldehyde in 0.1 M phosphate buffer, before being removed and prepared for vibratome sectioning. Floating sections were incubated at RT in 10% goat serum in PBS, before standard IHC procedure. Primary antibody was incubated at 1:5000 for 48 hours, goat anti-rabbit secondary was subsequently added for 1 hour after washing with PBS. Light microscopy staining was achieved with standard biotin-streptavidin/HRP procedure and DAB chromogen. The section was then mounted on slides with permount.
Quantity / Volume: 100 µL
State: Lyophilized Whole Serum
Reacts With: Mouse, Rat
Availability: In Stock
Alternate Names: Proto-oncogene c-Fos; Cellular oncogene; G0/G1 switch regulatory protein 7; Proto-oncogene protein; G0S7; p55; AP-1; FBJ murine osteosarcoma viral oncogene homolog, anti-CFOS
07/24/2017 - 01:49:36 PM
Staining Looks Great
Fluorescent immunohistochemistry on free-floating sections.
40 micron sections
1.Wash sections in 1X PBS for 3X5 min.
2.Incubate in PBS with 1% Triton-X 100 for 1 hour at RT.
3.Wash in PBS 3X5 min.
4.Incubate for 1 hour at RT in BLOCK.
5.Incubate with primary antibody 3 days in BLOCK. (In fridge)
6.Wash in PBS 3X5 min.
7.Incubate in secondary antibody overnight in BLOCK. (covered in foil in fridge)
8.Wash in PBS 3X5 min. (covered in foil)
9.Mount. Keep slides covered in foil until dry.
10.Coverslip. Use 140ul Vectashield on tissue, and return to foil for storage in fridge.
2% serum of secondary antibody(goat)
0.2% Triton X-100
Anti-C-Fos (Rabbit): 1:1000
Anti-rabbit 568 (red) (Alexa Fluor, abcam) 1:100
Anti-rabbit 488 (green) (Alexa Fluor, abcam) 1:100
Graduate Student: Rheall Roquet
Principal Investigator: Marie Monfils
University of Texas at Austin
05/24/2017 - 03:04:06 PM
Fluorescent immunohistochemistry on free-floating forebrain and brainstem sections.
Transcardially perfused rats: heparinized (50 U/ml) 0.01 M cold PBS or DMEM (~ 150 ml), then 4% PFA (~ 200 – 250 ml); post-fixed overnight (4% PFA); cryoprotected with 30% sucrose in 0.01M PBS (3 – 5 days); sectioned (35 microns) on a cryostat.
Sections rinsed (0.01M PBS) and blocked in 10% normal donkey serum (NDS) in 0.01M PBS (30 min)
Primary incubation: Overnight on shaker: Room temperature, free-floating sections, 1:3000 dilution of rabbit anti-C-Fos in 0.01 M PBS with 3% NDS and 0.3% Triton.
Secondary: 2 hrs, room temperature, donkey anti-rabbit IgG Alexa-488 (Jackson laboratories). 1:200 dilution in 0.01M PBS with 3% NDS.
Mounted: Gel coated slides; dried; coverslipped using Prolong Gold
Staining: Trial with hypertonic saline treated rat: Defined round nucei, with evident nucleolus in activated cells in the hypothalamic paraventricular nucleus. Good contrast, no background staining.
University of Missouri,
Columbia, Missouri, 65211
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Q & A Wall
We are looking at CFOS activation following a behavior task in rats to look at level of activation in different brain regions between different conditions. Will your antibody only work if the rats are injected with hypertonic saline? That sounds like it will give us inaccurate activation information.
Question By Emily B
cFos expession can be induced by a variety of means: (drugs, ie receptor agonists [we have got responses to 5HT1a, 5HT2a, 5HT2c, 5HT6 and 5HT agonists; there are other neurotransmitter agonists reported that work as well]; hormones (eg a number of reports on icv injected angiotensin II, CCK), physiological stimuli (tactile stimuli, pain, light, sound, epileptic loci, hypertonic saline.
Each stimulus for cfos expression results in cell nuclei localization, but each also has anatomically distinct distribution in the brain. cfos can be seen as a common pathway of many different stimuli. Its role in the nucleus is to promote gene expression of probably 100s of things that an activated cell needs to have to respond.
Hypertonic saline is a useful stressor (hyperosmotic stress) that is an easy, convenient method to induce cfos in a preceise location, magnocellular paraventricular and supraoptic neurons. In fact so much has been done using this stimulus of cfos expression that its primary use now is to validate cfos immunohistochemistry. If this doesnt work, nothing will. Its proper application is time dependent, max response time is 60-90 minutes. So work out the immunohistochemistry parameters in hypertonic saline injected animals and apply those ICC conditions to what you are really interested in studying.
Answer By Dr. Mark Brownfield on 2015-11-02 14:17:09
I usually dissolve my nicotine in physiological saline and I have been getting c-Fos labeling before. Do you have literature suggesting that hypertonic saline is better?
Question By Ozra D.
Injection of intraperitoneal hypertonic saline to provoke fos expression is most usually used to set ICC staining parameters. It is critical that tissue is harvested 60-90 minutes post injection for this and any other stress that is used.
Inject 1 ml/100 g body weight 1.5 M NaCl intraperitoneally. Harvest brains 90 minutes post injection. Time is critical.
Answer By Mark B. on 2015-10-02 09:35:19
This product contains the preservative sodium azide. The concentration percent of the sodium azide is ≤ .09%. Although this hazardous substance is a concentration below that required for the preparation of a Material Safety Data Sheet, we created a standard MSDS for your records.Download MSDS