| Storage and Preparation of the Antibody You will receive the Antibody to Fluoro-Gold in a small vial. It is a polyclonal antibody raised in rabbit. The vial will contain approximay 100µl of the antibody solution. It should be immediay frozen and stored in a cold （-20 degrees C）, dark, dry environment. The freezer section, without the frost free feature, of a good commercial refrigerator should suffice. If properly and continuously frozen, the antibody solution can be stored up to one year.
The antibody solution should remain frozen until ready for use. The 100µl aliquot of the Antibody is at a dilution of 1/100. The Antibody can be further diluted 1/50,000 to 1/100,000 times in BSA diluent （50 mM KPBS, 0.4% Triton, 1% BSA, 1% NGS） to produce 50ml to 100ml of working titer. This means you can dilute the solution you receive by 500 to 1,000 times. This should treat 300 to 1000 sections if you use the Vector elite kit. After preparation, the antibody solution can be stored for up to seven days in a cool （4 degrees C）, dark and dry environment. The refrigerator section of a good commercial refrigerator should suffice. We do not recommend that the solution be used beyond seven days after preparation. Do not refreeze the antibody solution.
Use of the Antibody
Fluoro-Gold can be injected using several different methods, including pressure, iontophoretic and other applications developed by a variety of researchers. See Schmued and Fallon, Fluoro-Gold: "A fluorescent retrograde axonal tracer with numerous unique properties," Brain Research, 377 （1986） 147-154 as well as Pieribone and Aston-Jones, "The Iontophoretic Application of Fluoro-Gold for the study of afferents to deep brain nuclei," Brain Research, 475 （1988） 259-271. Many researchers have developed their own modified procedures. Use of the antibody should not be dependent upon the methodology used to employ Fluoro-Gold.
After the Fluoro-Gold has been injected, floating sections （we used thirty um sections from a rat perfused with 4% formaldehyde） are incubated with the Fluoro-Gold Antibody solution overnight at 4 degrees C. Sections are washed, then incubated in Biotinylated GAR （Vector Labs） at 1/1000 for 1 hour at room temperature and washed again. Sections are then incubated in Avidin/Biotin （Vector Labs） at 1/1000 for 1 hour, washed and transferred to Diaminobenzidine （.04%） and Nickel Chloride （2.5%） in 0.1 M NaAcetate with 0.06% H2O2 for six minutes. Sections are then washed, mounted, dried, dehydrated and cover slipped.
It has been our experience that if stored and prepared in the manner set out above, each vial of the antibody should treat 300 to 1000 thirty um sections of the albino rat brain （or similar sized animals） if you use the Vector elite kit. However, you should experiment with the concentration and procedures to determine which best fits your circumstance.
Ki-67 Antibody-W Use Guide and Protocol
General:
Ki-67 Antibody-W reacts against the Ki-67 protein. The Ki-67 protein is an intrinsic marker of ongoing cell proliferation and is present during all active phases of the cell cycle （G1, S, G2 and mitosis） and is absent from resting cells （G0）. This means it is an excellent marker for determining the growth fraction of a given cell population. It is used extensively in diagnostic, research and drug discovery applications. Ki-67 Antibody-W is polyclonal and was raised in rabbit to the human peptide sequence –TPKEKAQALEDLAGFKELFQT – that was coupled to thyroglobulin by glutaraldehyde before injection into the rabbit. Our antibody has been used for immunocytochemistry in rat and human brain tissue. It will be sold with filter paper absorbed with the specific peptide for blocking. This allows the researcher to confirm the specific signal.
Characterization of the Ki-67 Antibody-W  | | A. Ki-67 positive cells in the hippocampus （10X） of a 7 day old rat | | B. Ki-67 cells in hippocampus （60X） taken on a confocal microscope | | C. Ki-67 block in hippocampus （10X） |
Specificity was ascertained by performing a blocking study. Adjacent sections from the hippocampus of a 7 day old rat were incubated with the Ki-67 Antibody-W alone （A-10X） and （B-60X） or with the Ki-67 Antibody-W preincubated with Ki-67 peptide （C-10X）. Note labeling in A and B and the absence of labeling in C, indicating specificity of the antibody binding.
Storage and Preparation:
The vial you will receive contains approximay 200µl of solution at a dilution of 1/100. The Ki-67Antibody-W can be further diluted to at least 1/20,000 in BSA diluent （50mM KPBS, 0.4% Triton, 1% BSA, 1% NGS）.
The antibody should be stored at -20C. Also included is filter paper absorbed with the specific peptide for blocking. 500µl of the diluted antibody can be added to the tube to achieve a 10µM block. Incubate for at least 1 hour at room temperature （RT） before applying to tissue. The antibody blocks specifically with this peptide and does not block with other peptides absorbed on filter paper.
Protocol:
Fresh frozen 10-30 micron sections are fixed in 4% paraformaldehyde （FA） for 1 hour at RT, washed in phosphate buffered saline （PBS）, and then treated in 10% Sodium Citrate for 40 minutes at 90C. The sections are allowed to cool for 20 minutes in the sodium citrate solution, washed in PBS and incubated with 0.1 to 0.3% H2O2 in PBS for 10 to 30 minutes. The tissue is again washed in PBS, then treated with an Avidin/Biotin blocking kit （Vector-cat #SP-2001）, washed and incubated with BSA diluent for 1 hour at RT. The Ki67 antibody （1/20,000） is applied to the tissue overnight at RT. After washing in PBS the sections are incubated in Biotinylated GAR at 1/1000 （Vector labs-cat#BA1000） for 1hr at RT, washed and then incubated in Avidin/Biotin at 1/1000 （Vector labs-cat#PK6100） for 1hr at RT. After washing the signal is visualized with Diaminobenzidine （0.04%） in 0.1M Sodium Acetate with 0.06% H2O2 for six minutes （Nickel Chloride at 2.5% may also be added）. Sections are then water washed （counterstained if desired）, dehydrated and cover slipped.
Ki-67 Antibody-W also works with the protocol used to visualize the BRDU Antibody-W sold by Fluorochrome, LLC. However, 4% FA perfused tissue may be used instead of fresh frozen sections.
Publications:
Perez et al, The Journal of Neuroscience, May 13, 2009:29（19）:6379-6387 Garcia-Fuster et al, in preparation: Individual differences in adult hippocampal neurogensis following experimenter administration of cocaine. | | |
GFAP Antibody-W Use Guide and Protocol
General:
GFAP Antibody-W reacts against the Glial Fibrillary Acidic Protein （GFAP）, which is a class-III intermediate filament and the main constitute of intermediate filaments in astrocytes and serves as a cell specific marker for mature glial cells. It is useful as a marker of neural stem cells and astrocytic cells, including many types of brain tumor derived from astrocytic cells, which express GFAP. GFAP Antibody-W is polyclonal and was raised in rabbit to the peptide sequence NAGFKETRASERAE （human, rat and mouse） coupled to thyroglobulin by glutaraldehyde before injection into the rabbit. Our antibody has been used for immunocytochemistry in rat and human brain tissue. It will also be sold with filter paper absorbed with the specific peptide for blocking. This allows the researcher to confirm the specific signal.
Characterization of the GFAP Antibody-W
|  | | A. GFAP glail cells in hippocampus （10X） of an adult rat | | B. GFAP glail cells in hippocampus （60X） taken on a confocal microscope | | C. GFAP block in hippocampus （10） |
Specificity was ascertained by performing a blocking study. Adjacent sections from the hippocampus of an adult rat were incubated with the GFAP Antibody-W alone （A-10X） and （B-60X） or with GFAP Antibody-W preincubated with GFAP peptide （C-10X）. Note labeling in A and B in glial cells and the absence of labeling in C, indicating specificity of the antibody binding.
Storage and Preparation:
The vial you will receive contains approximay200µl at a dilution of 1/100. The antibody can be further diluted to at least 1/30,000 in BSA diluent （50mM KPBS, 0.4% Triton, 1% BSA, 1% NGS）.
The antibody should be stored at -20C. Also included is filter paper absorbed with the specific peptide for blocking. 500µl of the diluted antibody can be added to the tube to achieve a 10 µM block. Incubate for at least 1 hour at room temperature （RT） before applying to tissue. The antibody blocks specifically with this peptide and does not block with other peptides absorbed on filter paper.
Protocol:
Fresh frozen 10-30 micron sections are fixed in 4% paraformaldehyde （FA） for 1 hour at RT, washed in phosphate buffered saline （PBS） and incubated with 0.1 to 0.3% H2O2 in PBS for 10 to 30 minutes. The tissue is again washed in PBS, then treated with an Avidin/Biotin blocking kit （Vector-cat #SP-2001）, washed and incubated with BSA diluent for 1 hr at RT. The GFAP Antibody-W （1/30,000） is applied to the tissue overnight at RT. After washing in PBS the sections are incubated in Biotinylated GAR at 1/1000 （Vector labs-cat#BA1000） for 1hr at RT, washed and then incubated in Avidin/Biotin at 1/1000 （Vector labs-cat#PK6100） for 1hr at RT. After washing the signal is visualized with Diaminobenzidine （0.04%） in 0.1M Sodium Acetate with 0.06% H2O2 for six minutes （Nickel Chloride at 2.5% may also be added）. Sections are then water washed （counterstained if desired）, dehydrated and cover slipped.
GFAP Antibody-W also works with the protocol used to visualize the BRDU Antibody-W sold by Fluorochrome, LLC. However, 4% FA perfused tissue may be used instead of fresh frozen sections.
| |
BrdU Antibody-W Use Guide and Protocol
General:
This antibody reacts against the synthetic thymidine analog BrdU, which when injected into an animal is incorporated during the S phase of the cell cycle into newly synthesized DNA strands of actively proliferating cells. The number of cells that survive can be measured by immunocytochemistry. As an outstanding tool to assess the proliferation state of a group of cells, it has become vital to drug discovery research, especially with cancer therapeutics and ascertaining the health of cells during ADME/Tox studies. BrdU Antibody-W is a polyclonal raised in rabbit against BrdU conjugated to bovine serum albumin （BSA）. Our antibody has been used for immunocytochemistry in rat brain tissue. It will be sold with filter paper absorbed with BrdU for blocking. This allows the researcher to confirm the specific signal.
Characterization of the BrdU Antibody-W
|  | | A. BrdU positive cells in dentate gyrus （60X） of an adult rat previously injected with BrdU. Photo taken on confocal microscope | | B. BrdU block in dentate gyrus |
Specificity was ascertained by performing a blocking study. Adjacent sections from the hippocampus of an adult rat that had been previously injected with BrdU were incubated with the BrdU Antibody-W alone （A-60X） or with BrdU Antibody-W preincubated with BrdU （B-60X）. Note labeling in A and the absence of labeling in B, indicating specificity of the antibody blocking.
Storage and Preparation:
The vial you will receive contains approximay 200µl at a dilution of 1/100. BrdU Antibody-W can be further diluted to at least 1/30,000 in BSA diluent （50mM KPBS, 0.4% Triton, 1% BSA, 1% NGS）.
The antibody should be stored at -20C. Also included is filter paper absorbed with BrdU for blocking. 500µl of the diluted antibody can be added to the tube to achieve a 10 µM block. Incubate for at least 1hour at room temperature （RT） before applying to tissue. The antibody blocks specifically with BrdU and does not block with other peptides absorbed on filter paper.
Protocol:
Fresh frozen 10-30 micron sections are fixed in 4% paraformaldehyde （FA） for 1 hour at RT, washed in phosphate buffered saline （PBS）, then treated with 50% Formamide/2X SSC （300 mM sodium chloride and 30 mM sodium citrate） at 65C for 2 hours. The sections are washed in 2X SSC, incubated in 2N HCL at 37C for 30 minutes, and then placed directly in 100mM Sodium Borate （pH 8.5） for 10 minutes at RT. The sections are washed in PBS and incubated with 0.1 to 0.3% H2O2 in PBS for 10 to 30 minutes. The tissue is again washed in PBS, then treated with an Avidin/Biotin blocking kit （Vector-cat #SP-2001）, washed and incubated with BSA diluent for 1 hr at RT. The BrdU Antibody-W （1/30,000） is applied to the tissue overnight at RT. After washing in PBS the sections are incubated in Biotinylated GAR at 1/1000 （Vector labs-cat#BA1000） for 1hr at RT, washed and then incubated in Avidin/Biotin at 1/1000 （Vector labs-cat#PK6100） for 1hr at RT. After washing the signal is visualized with Diaminobenzidine （0.04%） in 0.1M Sodium Acetate with 0.06% H2O2 for six minutes （Nickel Chloride at 2.5% may also be added）. Sections are then water washed （counterstained if desired）, dehydrated and cover slipped.
Publications:
Garcia-Fuster et al, in preparation: Individual differences in adult hippocampal neurogensis following experimenter administration of cocaine. | Notice: The original and only true Fluoro-Gold （Fluorogold） is produced by Fluorochrome, LLC and marketed by Fluorochrome, LLC and Histo-Chem Inc. Fluoro-Gold （Fluorogold） is an exclusive product of Fluorochrome, LLC. It has been sold by Fluorochrome and widely used since 1985. Other companies are marketing a product they claim is the same as or equivalent to Fluoro-Gold. In fact, the chemical structures of these compounds seem to be different from Fluoro-Gold. Certain physical properties of the compounds may be very different.
*CAUTION: Fluoro-Gold, Antibody to Fluoro-Gold and Fluoro-Ruby are for investigational use only in laboratory research animals or for tests in vitro. NOT FOR USE IN HUMANS. These drugs should be used only by persons regularly engaged in conducting neuroanatomical studies and tests in vitro or in animals used only for laboratory research. |
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