Goat anti-Rabbit IgG (Heavy & Light Chain) Antibody (FITC)

Details for Product No. ABIN101988, Supplier: Log in to see
Antigen
Epitope
Heavy & Light Chain
5027
2578
1931
1069
1007
857
330
299
35
34
31
8
7
7
3
3
2
2
1
1
1
1
1
1
Reactivity
Rabbit
2912
2859
2404
1752
1485
796
623
442
381
380
355
340
307
293
208
148
139
114
41
29
27
25
12
11
9
8
6
5
5
5
4
4
4
4
4
4
4
4
3
3
3
2
2
1
1
1
1
1
Host
Goat
6892
4912
1878
895
703
401
92
54
52
21
9
8
5
5
2
1
1
Clonality
Polyclonal
Conjugate
FITC
2235
1888
1740
1374
765
679
519
300
299
288
274
268
217
188
178
124
103
79
69
60
54
46
43
40
40
36
34
32
29
29
27
27
27
27
27
27
27
27
22
22
19
19
19
19
16
15
15
15
15
15
14
12
12
11
10
10
9
9
9
9
9
9
9
8
8
8
8
7
7
6
6
6
6
6
6
5
5
5
5
5
4
4
4
4
4
4
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
Application
FLISA, Immunomicroscopy (IM), Western Blotting (WB)
Options
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'Independent Validation' Badge
Antigen Rabbit IgG (Heavy & Light Chain)
Lot Number 611-1202
Method validated Immunofluorescence
Positive Control Lab stock CBD-SNAP antibody
Negative Control No SNAP-tag antibody
Notes We validate the specificity of the secondary goat anti-rabbit IgG (heavy & light chain) antibody (FITC) ABIN101988 for rabbit IgG antibody.
Primary Antibody ABIN1573927
Secondary Antibody ABIN101988
Protocol
  • Oyster visceral mass tissue is dissected and fixed in 4% paraformaldehyde in seawater overnight.
  • Serial dehydration process using an automated ASP300S Enclosed Tissue Processor (Leica Biosystems) as follows:
    • 70% ethanol for 45min
    • 90% ethanol for 45min
    • 90% ethanol for 45min
    • 100% ethanol twice for 45min
    • xylene twice for 45min
    • paraffin wax at 58°C 3 times for 30 min
  • Tissue is mounted in a paraffin block and hardened overnight before.
  • 8µm tissue sections are retrieved from the block and collected on circular glass cover slips.
  • Heat cover slips at 60°C for 1h.
  • Deparaffination and rehydration:
    • Xylene twice for 15min
    • 100% ethanol twice for 10min
    • 95% ethanol for 10 min
    • 85% ethanol for 10 min
    • 70% ethanol for 10 min
    • 50% ethanol for 10 min
    • 30% ethanol for 10 min
    • distilled water for 10 min
    • PBS for 10 min
  • Wash tissue sections with PBS with 0.05% triton X twice for 30min.
  • Permeabilize in PBS with 0.05% triton X overnight.
  • Treatment of the tissue sections with 1mg/mL sodium borohydride in PBS three times for 5min to reduce autofluorescence.
  • Wash sections in PBS 3 times for 15 min for at RT.
  • Block sections in PBST with 1% BSA for 2 hours at RT.
  • Incubate sections with CBD-SNAP antibody (lab stock) diluted 1:200 in PBST with 1% BSA overnight at 4°C to detect the location of chitin.
  • Wash sections in PBS 3 times for 15min with PBS at RT.
  • Additionally, incubate the CBD-SNAP and SNAP-tag double-stained sections with rabbit anti-SNAP antibody (antibodies-online, ABIN1573927, lot 13D000621) diluted 1:200 in PBST with 1% BSA overnight at 4°C.
  • Wash sections in PBS 3 times for 15min with PBS at RT.
  • Incubate sections with the secondary goat anti-rabbit IgG (heavy & light chain) antibody (FITC) (antibodies-online, ABIN101988, lot 611-1202) diluted 1:400 in PBST with 1% BSA for 2h at °C.
  • Wash sections in PBS three times for 15min at RT.
  • Counterstain with 0.1µg/mL DAPI in PBS for 15min at RT.
  • Wash sections in PBS three times for 15min at RT.
  • Mount sections on a microscopic slide using 50% glycerol in PBS.
  • Seal cover slips with nail polish.
  • Confocal imaging on Leica SPE.
  • Visualization of the data performed on LAS 3D software.
Experimental Notes To validate the specificity of the anti-rabbit FITC secondary antibody ABIN101988, 8µm paraffin sections of oyster visceral mass were observed in this study. We compared the fluorescence signals with immunofluorescence study. The negative control specimen was always compared with the test specimen or the positive control specimen on the same day, using the same laser power, gain, offset, accumulation/averaging settings on the Leica SPE confocal microscope. Visualization of the data was performed on LAS 3D software, with the same visualization setting to compare signal brightness. We found that the samples treated with anti-rabbit FITC secondary antibody ABIN101988 had similar fluorescence signals as the positive control Anti Rabbit Alexa 488. Excitation at the same laser wavelength and power did not generate fluorescence in the negative control section, when anti-rabbit FITC secondary antibody was applied in the absence of rabbit produced anti-SNAP antibody.
Validation Images
Immunofluorescence validation image for Goat anti-Rabbit IgG (Heavy & Light Chain) antibody (FITC) (ABIN101988) Immunofluorescence images of oyster visceral mass tissue, with the specificity of Ant...
Immunogen Rabbit IgG whole molecule
Isotype IgG
Specificity IgG (H&L)
Characteristics Concentration Definition: by UV absorbance at 280 nm
Research Area Immunology, Secondary Antibodies
Application Notes This product is designed for immunofluorescence microscopy, fluorescence based plate assays (FLISA) and fluorescent western blotting. This product is also suitable for multiplex analysis, including multicolor imaging, utilizing various commercial platforms.
Comment

Excitation/Emission wavelength: 494 nm/514 nm

Restrictions For Research Use only
Format Lyophilized
Reconstitution Restore with deionized water (or equivalent)
Concentration 2.0 mg/mL
Buffer 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
Preservative Sodium azide
Precaution of Use WARNING: Reagents contain sodium azide. Sodium azide is very toxic if ingested or inhaled. Avoid contact with skin, eyes, or clothing. Wear eye or face protection when handling. If skin or eye contact occurs, wash with copious amounts of water. If ingested or inhaled, contact a physician immediately. Sodium azide yields toxic hydrazoic acid under acidic conditions. Dilute azide-containing compounds in running water before discarding to avoid accumulation of potentially explosive deposits in lead or copper plumbing.
Handling Advice Product is photosensitive and should be protected from light.
Supplier Images
Western Blotting (WB) image for Goat anti-Rabbit IgG (Heavy & Light Chain) antibody (FITC) (ABIN101988) FITC (fluorescein) and HRP (horse radish peroxidase) conjugated secondary antibody wa...
Background publications Blakeslee, Baines: "Immunofluorescence using dichlorotriazinylaminofluorescein (DTAF). I. Preparation and fractionation of labelled IgG." in: Journal of immunological methods, Vol. 13, Issue 3-4, pp. 305-20, 1977 (PubMed).