Plasmodium falciparum (FVO) were cultured in vitro until parasitaemia reached ~5 to 10% and blood smears were prepared. Following fixation in cold acetone, selected plasma samples were diluted 1:50 and ~15 μl was applied to the smears for 30 min at RT. Slides were washed with PBS and incubated with 15 μl of a 1:100 dilution of fluorescein isothiocynate (FITC)-labelled anti-human IgG (AffiniPure F(ab’) Fragment Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch Lab, Inc., Cat# 109-096-098) or anti-human IgM (rabbit anti-human IgM (Fc specific, Sigma, Cat # 14121 followed by FITC-AffiniPure F(ab’) Fragment Goat Anti-Rabbit IgG (H + L), Jackson ImmunoResearch Lab, Inc., Cat# 111-096-003). MAb 2G12 and IE1 and normal mouse plasma were used as a positive and negative controls, respectively, because the MAb give a distinct pattern of fluorescence when bound to HRP2. After 30 min, smears were washed, mounted in mounting medium (Barbital buffer, pH 8.5 in 50% glycerol), and examined using a Zeiss Axiovision HBO 100 fluorescent microscope.
Affinipure f ab fragment goat anti rabbit igg h l
Manufactured by Jackson ImmunoResearch
Sourced in United States
AffiniPure F(ab') Fragment Goat Anti-Rabbit IgG (H + L) is a laboratory product manufactured by Jackson ImmunoResearch. It is a purified fragment of goat antibodies that specifically bind to the Fab region of rabbit immunoglobulin G (IgG) molecules.
Automatically generated - may contain errors
Lab products found in correlation
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Rabbit anti olfm4, by Cell Signaling Technology (1 mentions)
Goat anti gfp, by Rockland Immunochemicals (1 mentions)
Alexa fluor 488 or 594, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Mouse anti ki67, by BD (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Anti akt 9272, by Cell Signaling Technology (1 mentions)
Na931v, by GE Healthcare (1 mentions)
Anti phospho akt ser473 9271, by Cell Signaling Technology (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
4 protocols using affinipure f ab fragment goat anti rabbit igg h l
1
Immunofluorescent Detection of Plasmodium falciparum
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Plasmodium falciparum (FVO) were cultured in vitro until parasitaemia reached ~5 to 10% and blood smears were prepared. Following fixation in cold acetone, selected plasma samples were diluted 1:50 and ~15 μl was applied to the smears for 30 min at RT. Slides were washed with PBS and incubated with 15 μl of a 1:100 dilution of fluorescein isothiocynate (FITC)-labelled anti-human IgG (AffiniPure F(ab’) Fragment Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch Lab, Inc., Cat# 109-096-098) or anti-human IgM (rabbit anti-human IgM (Fc specific, Sigma, Cat # 14121 followed by FITC-AffiniPure F(ab’) Fragment Goat Anti-Rabbit IgG (H + L), Jackson ImmunoResearch Lab, Inc., Cat# 111-096-003). MAb 2G12 and IE1 and normal mouse plasma were used as a positive and negative controls, respectively, because the MAb give a distinct pattern of fluorescence when bound to HRP2. After 30 min, smears were washed, mounted in mounting medium (Barbital buffer, pH 8.5 in 50% glycerol), and examined using a Zeiss Axiovision HBO 100 fluorescent microscope.
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Plasmodium falciparum (FVO) were cultured in vitro until parasitaemia reached ~5 to 10% and blood smears were prepared. Following fixation in cold acetone, selected plasma samples were diluted 1:50 and ~15 μl was applied to the smears for 30 min at RT. Slides were washed with PBS and incubated with 15 μl of a 1:100 dilution of fluorescein isothiocynate (FITC)-labelled anti-human IgG (AffiniPure F(ab’) Fragment Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch Lab, Inc., Cat# 109-096-098) or anti-human IgM (rabbit anti-human IgM (Fc specific, Sigma, Cat # 14121 followed by FITC-AffiniPure F(ab’) Fragment Goat Anti-Rabbit IgG (H + L), Jackson ImmunoResearch Lab, Inc., Cat# 111-096-003). MAb 2G12 and IE1 and normal mouse plasma were used as a positive and negative controls, respectively, because the MAb give a distinct pattern of fluorescence when bound to HRP2. After 30 min, smears were washed, mounted in mounting medium (Barbital buffer, pH 8.5 in 50% glycerol), and examined using a Zeiss Axiovision HBO 100 fluorescent microscope.
2
Immunofluorescence Staining of Cell Markers
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Secondary antibodies used are goat anti-rabbit or mouse AlexaFluor 488 or 594, donkey anti-goat or rabbit AlexaFluor 488 or 594 (all from Invitrogen). Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma, D8417). Primary antibodies used are rabbit anti-active CASPASE 3 (Cell Signaling, 9661), mouse anti-KI67 (BD Pharmingen, 550609), rabbit anti-OLFM4 (Cell Signaling, 39141), goat anti-PTBP1 (Santa Cruz sc-16547), rabbit anti-PTBP1 (gift from Dr. Douglas Black), goat anti-GFP (Rockland, 600-101-215), rabbit anti-P53 (Leica Biosystems, NCL-L-p53-CM5p), rabbit anti-PHLDA3 (LifeSpan BioSciences, LS-C499531-100), rabbit anti-Lysozyme antibody (Diagnostic Biosystems, RP028), and Affinipure Fab Fragment Goat anti-Rabbit IgG (H + L) (Jackson ImmunoResearch, 111-007-003).
3
Dual Immunolabeling of PR, POMC, and NPY
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Double staining of PR with POMC or NPY was performed on sections adjacent to those with single POMC/NPY neuronal staining throughout the INF. In brief, sections after PR-DAB-Ni development were incubated with 20 µg/mL AffiniPure Fab fragment goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, West Grove, PA, USA) then incubated with the POMC/NPY antibody, followed by a secondary antibody and ABC incubation, and DAB development.
4
Antibody Validation and Gene Expression Profiling
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We used the following primary antibodies as follows: anti-Akt (9272, Cell Signaling) at 1:1000; anti-phospho(Ser473)-Akt (9271, Cell Signaling) at 1:1000; anti-cFos (PC38, Calbiochem) at 1:10000; anti-Darpp32 (AB10518, Millipore) at 1:2500; anti-ERα (06–935, Millipore) at 1:500; anti-Parvalbumin (PVG214, Swant) at 1:5000. In the immunostaining procedure, we used secondary antibodies anti-rabbit IgG and anti-goat IgG conjugated with Alexa Fluor 488 or Alexa Fluor 568 at 1:1000 (Invitrogen), AffiniPure Fab Fragment Goat Anti-rabbit IgG (H+L) (111-007-003, Jackson Laboratories). In the Western blot protocol, we used HRP-conjugated secondary antibodies anti-rabbit IgG (31460, Thermo Scientific) and anti-mouse IgG (NA931V, GE Healthcare) at 1:10000. The specificity of each antibody used in this study has been extensively tested by other groups and showed specific and expected staining in our hands.
For quantitative PCR, we used the following TaqMan probes (from Thermofisher): Gdnf1-2 (Gdnf exons 1 to 2), Mm00599849_m1; Pvalb (parvalbumin), Mm00443100_m1; Actb (βActin), Mm00443100_m1; Gapdh, Mm99999915_g1; Hmbs, Mm00660262_g1.
17β-estradiol (E8875, Sigma) was used to make up the SILASTIC implants, and β-estradiol 3-benzoate (E-8515, Sigma) for subcutaneous injection.
For quantitative PCR, we used the following TaqMan probes (from Thermofisher): Gdnf1-2 (Gdnf exons 1 to 2), Mm00599849_m1; Pvalb (parvalbumin), Mm00443100_m1; Actb (βActin), Mm00443100_m1; Gapdh, Mm99999915_g1; Hmbs, Mm00660262_g1.
17β-estradiol (E8875, Sigma) was used to make up the SILASTIC implants, and β-estradiol 3-benzoate (E-8515, Sigma) for subcutaneous injection.
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