A11039

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The A11039 is a compact and versatile laboratory instrument designed for a range of analytical applications. It features high-performance capabilities and advanced technology to deliver accurate and reliable results. The core function of the A11039 is to facilitate precise measurement and analysis tasks within the laboratory environment.

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Lab products found in correlation

115 protocols using a11039

Immunofluorescence Staining of Microglia and Astrocytes

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Immunofluorescence staining for microglia (Iba1) and astrocytes (GFAP) was performed over 3 days, using the reagents described above. On the first day, heat-fixed, paraffin-embedded mouse tissue slides were dewaxed, rehydrated, and antigen retrieved at 60 °C for 30 min, and then blocked for 1 h at room temperature. Primary antibody solution contained 1:1000 rabbit anti-Iba1 (Wako Japan #019-19741) and 1:1000 chicken anti-GFAP antibodies in blocking solution. Blocking solution with no primary antibodies was used as negative control. After overnight incubation, the slides were rinsed three times in PBS and then blocked for 10 min before application of secondary antibodies. The secondary antibody solution contained 1:1000 AF488-conjugated goat anti-chicken (Invitrogen A-11039) and 1:1000 AF647-conjugated goat anti-rabbit antibodies (Invitrogen A-32733) in blocking solution. From this point onwards, care was taken to prevent exposure of the tissue to light. After overnight incubation, on the final day, tissues were rinsed in PBS and counterstained with 1:5000 DAPI, before being mounted and sealed with nail polish. Stained slides were stored at 4 °C until imaged.

Blackmore D.G., Turpin F., Palliyaguru T., Evans H.T., Chicoteau A., Lee W., Pelekanos M., Nguyen N., Song J., Sullivan R.K., Sah P., Bartlett P.F, & Götz J. (2021). Low-intensity ultrasound restores long-term potentiation and memory in senescent mice through pleiotropic mechanisms including NMDAR signaling. Molecular Psychiatry, 26(11), 6975-6991.

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Immunostaining of 3D Neurospheroids

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Immunostaining of 3D neurospheroids was performed as previously described ^{30 (link)}. Briefly, spheroids were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) overnight at 4°C, washed several times with PBS, and permeabilized in 0.2% Triton-X in PBS for 30 minutes at room temperature. All wash steps and solution changes were accomplished via a two-thirds solution exchange protocol (i.e., aspirate to 25μL/well, add 50μL/well fresh solution). Blocking solution (2% BSA + 0.2% Triton-X in PBS) was then added and spheroids were incubated at 37°C for >1 hour under gentle agitation. Primary antibodies (MAP2, Synaptic Systems 188 004, 1:1000; GFAP, Novus Biologicals, NBP1-05198, 1:1000) were added in blocking solution and incubated overnight at 37°C with gentle agitation followed by 6 washes using PBS + 0.2% Triton-X. Spheroids were blocked again for >1 hour and incubated with secondary antibodies (Invitrogen, A-21450 and A-11039) diluted 1:500 in blocking solution with DAPI (Invitrogen, 1:5000) overnight at 37°C with gentle agitation, protected from light. Spheroids were then washed 6 times using PBS, transferred to flat bottom plates and either stored at 4°C or imaged using an Operetta CLS spinning disk confocal microscope (Perkin Elmer) at 20x magnification (Fig. S1).

Boutin M.E., Strong C.E., Van Hese B., Hu X., Itkin Z., Chen Y.C., LaCroix A., Gordon R., Guicherit O., Carromeu C., Kundu S., Lee E, & Ferrer M. (2022). A multiparametric calcium signal screening platform using iPSC-derived cortical neural spheroids. SLAS discovery : advancing life sciences R & D, 27(4), 209-218.

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Immunohistochemistry of Mouse Brain Slices

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Mice were transcardially perfused with 4% PFA (4% paraformaldehyte in 0.1 M phosphate buffer) and after perfusion, brains were sliced coronally (50 μm thick) with a vibratome and processed for immunostaining. Primary antibodies, including chicken polyclonal anti-GFP (Abcam AB13970, 1:1000), mouse monoclonal anti-GFP (Synaptic Systems, 132 011, 1:500), Chicken anti-RFP (Synaptic Systems, 409 006, 1:500), mouse anti-TetR Monoclonal Antibody (Clone 9G9, Takara, 63113, 1:500), mouse anti-NeuN (Chemicon, MAB377, 1:1000), rabbit anti-GFAP (Dako, Z0334, 1:500), rabbit anti-c-Fos (Cell Signaling, 9F6, #2250, 1:500), and rabbit anti-P2Y12 (AnaSpec, AS-55043A, 1:1000) and secondary antibodies, including goat anti-chicken 488 (Invitrogen, A11039, 1:2000), goat anti-mouse 488 (Invitrogen, A11029, 1:2000), goat anti-chicken 594 (Invitrogen, A11042, 1:2000), goat anti-rabbit 647 (Invitrogen, A21245, 1:2000) were used for immunostaining. Brain slices were incubated with 4’,6-diaminodino-2-phenylindole (DAPI, Invitrogen, 1:2000) for 10 min and washed with PBS three times before mounting onto slides. Immunostaining images were acquired by NIS-Elements AR (Nikon, v4.40.00) with a Nikon A1 Laser Scanning Confocal Microscope (LSCM). NIS-Elements AR Analysis (Nikon, v4.40.00) was used to analyze the confocal images.

Shen Y., Zhou M., Cai D., Filho D.A., Fernandes G., Cai Y., de Sousa A.F., Tian M., Kim N., Lee J., Necula D., Zhou C., Li S., Salinas S., Liu A., Kang X., Kamata M., Lavi A., Huang S., Silva T., Do Heo W, & Silva A.J. (2022). CCR5 closes the temporal window for memory linking. Nature, 606(7912), 146-152.

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Tissue Preparation and Immunostaining of Mouse Brain Sections

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Mice were perfused trans-cardially with PBS followed by 4% paraformaldehyde fixation solution. Brains were dissected and post-fixed in fixation solution at 4°C overnight, soaked in 30% sucrose solution overnight, embedded in O.C.T. (Tissue-Tek, Sakura Finetek USA, INC., Torrance, CA), frozen, and cut into 20–35 μm coronal sections. After washing, sections were blocked for 1 hr (3% normal goat serum in PBS, 0.4% Triton X-100, 0.2% sodium azide) followed by incubation with primary antibody: chicken anti GFP (abcam, ab13970), rabbit anti-Adcy3 (Santa Cruz Biotechnology, sc-588) or mouse anti FLAG M1 (Sigma, F3040) overnight at 4°C. Sections were extensively washed in PBS, and then incubated with secondary antibody: goat anti-chicken Alexa fluor 488 (Invitrogen, A11039), goat anti-mouse Alexa fluor 488 (Invitrogen, A11001), or goat anti-rabbit Alexa fluor 633 (Invitrogen, A21071).

Siljee J.E., Wang Y., Bernard A.A., Ersoy B.A., Zhang S., Marley A., Von Zastrow M., Reiter J.F, & Vaisse C. (2018). Subcellular localization of MC4R with ADCY3 at neuronal primary cilia underlies a common pathway for genetic predisposition to obesity. Nature genetics, 50(2), 180-185.

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Immunostaining of Drosophila Fat Body and Midgut

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Bisected third instar larvae were inverted and fixed with 3.7% paraformaldehyde in PBS overnight at 4 °C. Next, samples were rinsed twice and washed for 2 h in PBS, permeabilized for 15 min in PBTX-DOC (PBS with 0.1% Triton X-100 and 0.05% sodium deoxycholate) and blocked for 3 h in 3% goat serum in PBTX-DOC. Samples were then incubated overnight at 4 °C with primary antibodies in 1% goat serum in PBTX-DOC. After 3 × 30 min washes in PBTX-DOC, samples were incubated with secondary antibodies diluted 1:1,500 in 1% goat serum in PBTX-DOC for 4 h at room temperature. Finally, after 3 × 15 min washes in PBTX-DOC and 1 × 15 min in PBS, fat bodies and midguts were dissected and mounted in 50% glycerol/PBS with 0.2 μM DAPI. The following secondary antibodies were used: Alexa 488 anti-chicken, Alexa 488 anti-rabbit, Alexa 546 anti-rabbit, Alexa 568 anti-rat, Alexa 568 anti-mouse, Alexa 647 anti-rabbit (all used in 1:1,500, Invitrogen A11039, A11034, A11035, A11077, A1104, A21245, respectively).

Nagy P., Kárpáti M., Varga Á., Pircs K., Venkei Z., Takáts S., Varga K., Érdi B., Hegedűs K, & Juhász G. (2014). Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila. Autophagy, 10(3), 453-467.

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Immunofluorescent Detection of Co-expressed GFP

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A 1 in 3 series of sections adjacent to those used for β-galactosidase staining were processed to detect the co-expressed GFP reporter present in the tetO-lacZ-nls-GFP line. Sections were rinsed in TBS before non-specific binding was blocked with 5 % normal goat serum in TBS with 0.1 % Triton X-100 for 90 min. Sections were then incubated in chicken anti-GFP (1:500, Abcam, ab13970) for 48 h at 4 °C. Tissue was washed in TBS before being incubated for 90 min with an Alexa-488 conjugated goat anti-chicken IgG secondary antibody (1:500, Invitrogen, A-11039). Sections were washed in TBS, mounted onto Superfrost Plus slides, and coverslipped with Vectashield containing 1.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI).

Yetman M.J., Lillehaug S., Bjaalie J.G., Leergaard T.B, & Jankowsky J.L. (2015). Transgene expression in the Nop-tTA driver line is not inherently restricted to the entorhinal cortex. Brain structure & function, 221(4), 2231-2249.

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NRF2 Modulation in Nerve Injury

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Mice were first dosed daily with either sulforaphane (10 mg/kg, i.p.), exemestane (10 mg/kg, i.p.), or the appropriate vehicle for 2 days before surgical constriction of the maxillary nerve, after which they were dosed every 24 hours thereafter. NRF2 immunostaining was performed 24 hours after the fourth dose. The following antibodies were used: primary antibodies: rabbit anti-NRF2 (1:100) and chicken anti-MAP2 (1:5000); secondary antibodies: goat anti-rabbit (Alexa 568, A-11011, Invitrogen) and goat anti-chicken (Alexa 488, A-11039, Invitrogen).

Vasavda C., Xu R., Liew J., Kothari R., Dhindsa R.S., Semenza E.R., Paul B.D., Green D.P., Sabbagh M.F., Shin J.Y., Yang W., Snowman A.M., Albacarys L.K., Moghekar A., Pardo-Villamizar C.A., Luciano M., Huang J., Bettegowda C., Kwatra S.G., Dong X., Lim M, & Snyder S.H. (2022). Identification of the NRF2 transcriptional network as a therapeutic target for trigeminal neuropathic pain. Science Advances, 8(31), eabo5633.

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Quantifying Cell Types in Nephron Segments

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Determination of the numbers of each cell type per unit length in microdissected PTs and CCDs from UNx and Sham mice was carried out using immunocytochemistry employing antibodies recognizing cell type specific markers, based on Purkerson et al.^{79 (link)}. The primary antibodies used were rabbit anti-AQP1 (LL266, in house, 1:100), mouse anti V-ATPase B1/B2 (F-6, sc-55544, Santa Cruz Biotechnology, Santa Cruz, CA, 1:100), anti-chicken AQP2 (CC 265, in house, 1:1000) and Alexa Fluor 568 phalloidin (A12380, Invitrogen, 1:400). The secondary antibodies were Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 488 goat anti-chicken, Alexa Fluor 568 goat anti-chicken and Alexa Fluor M-594 goat anti-mouse IgG. (A11034, A11039, A11041 and A11032, Invitrogen) each at 1:400 dilution. Cell nuclei were labelled with DAPI. Confocal fluorescence images were recorded with a Zeiss LSM780 confocal microscope using a 20× objective lens by Z-stack scanning. 3D images are reconstructed using z-stack files, and cell counting was performed on three-dimensional reconstructed tubule images using IMARIS Scientific Image Processing & Analysis software (v7.7.1, Bitplane, Zurich, Switzerland). Counting was automated using IMARIS “spot analysis” for nuclei. Tubule volume was calculated using IMARIS “surface analysis”.

Kikuchi H., Chou C.L., Yang C.R., Chen L., Jung H.J., Park E., Limbutara K., Carter B., Yang Z.H., Kun J.F., Remaley A.T, & Knepper M.A. (2023). Signaling mechanisms in renal compensatory hypertrophy revealed by multi-omics. Nature Communications, 14, 3481.

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Immunohistochemical Labeling of Fluorescent Proteins

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YFP immunoreactivity was detected using chicken anti-GFP (1:500; Abcam). tdRFP expression was detected with an antibody raised against dsRed-Express (1:500; #632496, Takara Bio, USA). Free-floating sections were incubated for 48h at 4°C with primary antibodies in blocking solution (PBS with 0.3% triton X-100, 1%BSA, 1% Normal Goat Serum, 1% Normal Donkey Serum) followed by incubation with Alexa Fluor 488-conjugated goat anti-chicken antibody (1:1000; #A-11039, Invitrogen) and/or Alexa Fluor 568-conjugated donkey anti-rabbit antibody (1:1000; #A-10042, Invitrogen) in blocking solution for 2 h. Sections were subsequently mounted and coverslipped for microscopic analysis of fluorescent labeling.

Card J.P., Johnson A.L., Llewellyn‐Smith I.J., Zheng H., Anand R., Brierley D.I., Trapp S, & Rinaman L. (2018). GLP‐1 neurons form a local synaptic circuit within the rodent nucleus of the solitary tract. The Journal of Comparative Neurology, 526(14), 2149-2164.

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Immunohistochemical Staining of Mouse Brain

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Mice were euthanised by Avertin overdose and perfused with 4% paraformaldehyde. Brains were removed and placed in 4% paraformaldehyde for 24 h then stored in PBS at 4°C prior to sectioning. 50 µm coronal sections were obtained using a vibratome and stored at −20°C. At the time of staining, sections underwent three 10-minute PBS-T (PBS+ 0.2% Triton X-100) washes then incubated in blocking buffer (5% normal goat serum) for 1 h. Primary antibodies were applied overnight at 4°C: chicken anti-GFP from Invitrogen A10262 (1:1000 dilution) and rabbit anti-RFP from Rockland Immunochemicals 600-401-379(1:2000 dilution). Sections underwent three 10-minute washes in PBS-T then were incubated with secondary antibody solution (goat anti-chicken conjugated with Alexa Fluor 488 Invitrogen A11039 (1:200 dilution) and goat anti-rabbit conjugated with Alexa Fluor 568 Invitrogen A11011 (1:500 dilution)) for 2 hours. Sections underwent another three 10 minute washes prior to coverslip mounting using VECTASHIELD with DAPI.

Redondo R.L., Kim J., Arons A.L., Ramirez S., Liu X, & Tonegawa S. (2014). Bidirectional switch of the valence associated with a hippocampal contextual memory engram. Nature, 513(7518), 426-430.

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