Cell survival was assessed by DAPI nuclear staining. This method to distinguish physiologically intact from dead or dying neurons was validated by comparison with testing membrane integrity with the trypan blue exclusion assay [49 (link)] and TUNEL staining [this study]. Fixed cells were washed three times in PBS before washing twice with PBS/0.1% Triton-X-100 (PBST). Cultures were incubated for 30 min in DAPI (Sigma-Aldrich; Munich, Germany; 1:1000 in PBST) in the dark. Subsequently, coverslips were washed five times in PBS and once in DABCO (Roth, Kalsruhe, Germany) before mounting in DABCO. Experimental groups were evaluated with an epifluorescence microscope (Zeiss Axioskop; 40 × objective, Oberkochen, Germany) equipped with a Spot CCD camera (Invisitron, Puchheim, Germany). Two rows of non-overlapping photographs (~ 80 on average) covering the entire extension of the coverslips were taken from each cell culture for subsequent analysis. Live/Dead assessment was performed by manual counting of DAPI stained nuclei by an observer who was blinded with respect to the culture treatment. Cell counting was supported by using ImageJ Cell counter plug-in (Fiji ImageJ by NIH) as described elsewhere [46 (link)–48 (link)].
Dabco
Manufactured by Carl Roth
Sourced in Germany
DABCO is a chemical reagent commonly used as a catalyst and base in organic synthesis. It is a bicyclic compound with the chemical formula C6H12N2. DABCO is frequently employed in various organic reactions, such as ester and ether formations, as well as in the synthesis of heterocyclic compounds.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Hoechst, by Merck Group (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Mowiol, by Carl Roth (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
Keratin 14, by BioLegend (2 mentions)
Keratin 8, by Abcam (2 mentions)
Mowiol, by Zeiss (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Rat tail collagen, by Merck Group (1 mentions)
Axiovert 10, by Zeiss (1 mentions)
Mowiol, by Merck Group (1 mentions)
Epifluorescence microscope, by Zeiss (1 mentions)
Axioskop, by Zeiss (1 mentions)
A31572, by Thermo Fisher Scientific (1 mentions)
Ht501128 4l, by Merck Group (1 mentions)
P1379 100ml, by Merck Group (1 mentions)
Axio observer z1 inverted fluorescence microscope, by Zeiss (1 mentions)
Roti immunoblock, by Carl Roth (1 mentions)
11 protocols using dabco
1
DAPI-based Cell Viability Quantification
2
Immunofluorescent Staining of Virus-Infected CRFK Cells
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CRFK cells were cultured on sterile cover slips in 24 well plates pretreated with rat tail collagen (Sigma). Cells were mock or virus-infected and treated with 2 μg ml−1 tunicamycin 4 h p.i. At 15 h p.i. the cells were fixed with PBS containing 4% paraformaldehyde for 20 min at 4 °C, followed by three PBS washes. Cells were permeabilized with Triton X-100 (1% in PBS) for 5 min at room temperature. The cells were rinsed twice with PBS and residual binding sites were blocked with 1× Roti®-ImmunoBlock (Roth) for 10 min at room temperature. The cells were then incubated with primary antibodies (1:3 dilution) or sera (1:1500 dilution) in PBS for 1 h at 37 °C, followed by three PBS washes. Subsequently the cells were incubated with secondary antibodies conjugated to cyanogen-3 (1:500 dilution, Dianova) in PBS for an additional hour at 37 °C. The DNA was counterstained with 66 μg ml−1 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. After three washes the cover slips were mounted with Mowiol (Sigma) containing the anti-fading reagent DABCO (1,4-diazabicyclo(2.2.2)octane, Roth). The immunofluorescent staining was visualized by fluorescence microscopy (Axiovert 10, Zeiss).
3
Quantifying Cell Survival from Microscopy
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Both hemocyte and neuron survival was analyzed as described previously (Miljus et al., 2014 (link); Hahn et al., 2017 (link), 2019 (link)). After fixation, coverslips with attached cells were washed (5 min per step) three times in PBS followed by two wash steps in PBS/0,1% Triton-X-100 (PBST). Cells were stained with Dapi (1:1000 in PBST; Sigma-Aldrich; Munich, Germany) for 30 min in the dark. Subsequently, coverslips were washed five times in PBS before mounting on microscopy slides in DABCO (Roth, Karlsruhe, Germany).
Coverslips were imaged with an epifluorescence microscope (Zeiss Axioskop; Oberkochen, Germany; 40x objective was used for locust neurons or hemocytes, 63x oil objective was used for tribolium neurons) equipped with a Spot CCD camera (Invisitron, Puchheim, Germany). Non-overlapping series of photographs passing the center of the coverslip to the left and the right were taken from all cultures (~80 pictures per locust culture and ~120 pictures per tribolium culture). Cells were manually scored as intact or dead/dying on the basis of Dapi-fluorescence pattern reflecting nuclear chromatin structure. The scorer was blinded with respect to the culture treatment during counting. Cell counting was supported by ImageJ Cell counter plug-in (Fiji ImageJ by NIH) as described elsewhere (Miljus et al., 2014 (link); Hahn et al., 2019 (link); Knorr et al., 2020 (link)).
Coverslips were imaged with an epifluorescence microscope (Zeiss Axioskop; Oberkochen, Germany; 40x objective was used for locust neurons or hemocytes, 63x oil objective was used for tribolium neurons) equipped with a Spot CCD camera (Invisitron, Puchheim, Germany). Non-overlapping series of photographs passing the center of the coverslip to the left and the right were taken from all cultures (~80 pictures per locust culture and ~120 pictures per tribolium culture). Cells were manually scored as intact or dead/dying on the basis of Dapi-fluorescence pattern reflecting nuclear chromatin structure. The scorer was blinded with respect to the culture treatment during counting. Cell counting was supported by ImageJ Cell counter plug-in (Fiji ImageJ by NIH) as described elsewhere (Miljus et al., 2014 (link); Hahn et al., 2019 (link); Knorr et al., 2020 (link)).
4
Immunofluorescence Staining of Cryosections
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Cryosections were dried for 10 min at room temperature, fixed for 10 min in 10% formalin (#HT501128-4L, Sigma Aldrich, Germany). Embedding medium was removed with 0.02% Tween20 (T; #P1379-100ML, Sigma Aldrich, Germany) in tris buffered saline (TBS) twice for 5 min. All unspecific binding sites were blocked for 1 h at room temperature in 10% normal donkey serum (#S30-100ML, EMD Milipore, Germany), 1% bovine serum albumin (#0163.2, Roth, Germany) in TBST, followed by overnight incubation of the 1st antibody diluted as indicated in 1% bovine serum albumin in TBST at 4 °C. After 30 min at room temperature 1st antibody was carefully washed away with 4 repetitions of 5 min TBST. The 2nd antibody (A21247; A31571; A21434; A31572; A21432; Invitrogen and 703-545-155; Jackson ImmunoResearch) was diluted 1:500 in 1% bovine serum albumin in TBS, DAPI (300 nM, #D9542-5MG, Sigma Aldrich, Germany) was added, and after 1 h incubation at room temperature and 4 times 5 min wash in TBS, all sections were mounted with Dabco (#0718.1, Roth, Germany) in Mowiol (#0713.1 Roth, Germany). TUNEL staining (In Situ Cell Death Detection Kit, TMR red; #12156792910 Roche; Merck Germany) was applied according to the manual after 2nd antibody and before mounting with Dabco/Mowiol.
5
Immunocytochemical Visualization of PMEL
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For immunocytochemistry, MAC-T cells were seeded on coverslips. After overnight culture growth, coverslips were washed three times with PBS and fixed with ice-cold methanol (10 min, −20 °C). Anti-PMEL (1:100) primary antibody was incubated overnight at 4 °C. Unspecific antibody binding was blocked with Roti-Immunoblock (Carl Roth GmbH). Goat anti-rabbit IgG secondary antibody conjugated with Alexa Fluor® 488 (1:1000; # A11034, Life Technologies, Germany) was used to detect specific binding of the primary antibody. Nuclei were counterstained with 600 nM DAPI (Carl Roth GmbH). The same protocol, excluding incubation with the primary antibody, was used for the negative controls. Cells were mounted with DABCO (Carl Roth GmbH) and analyzed with the Axio Observer.Z1 inverted fluorescence microscope (Carl Zeiss Microscopy GmbH) using the Plan-Apochromat 63x/1.4 Oil DICIII objective.
6
Immunofluorescence Staining in Diverse Tissues
7
Multiparametric Immunofluorescence Staining
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mLN were extracted and fixed for 72 h in 4 % Formalin at RT. The formalin was changed every 24 h. The tissues were embedded in paraffin. Sections of 3 µm thickness were generated. Heat-induced antigen retrieval of tissue sections was performed in 20 mM citric acid buffer (pH 6.0). The following primary antibodies/conjugates were used: rabbit anti-CD3 (A0452 Dako), PNA-biotin (B-1075, VECTOR) and rat anti-Foxp3 (FJK-16s, ThermoFisher). For detection the following secondary antibodies were used: donkey anti-rat Alexa FluorTM 488, donkey anti-rabbit Alexa FluorTM 555, streptavidin Alexa FluorTM 647 all from ThermoFisher. The tissues were blocked with normal rat serum (NRS) (STEMCELL), in order to allow a third staining step with the rat anti-B220-AlexaFluor594 (RA3-6B2, Biolegend) antibody and Hoechst (Sigma-Aldrich). Tissues were mounted in Mowiol supplemented with DABCO (ROTH). Images were acquired at the Evos FL Auto 2 fluorescence microscope (ThermoFisher) and evaluated using the software Fiji (ImageJ) (45 (link)).
8
Epo and Splice Variant Effects on Cultured Cells
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Four-day-old primary cultures were maintained in L-15 medium supplemented with 0.5% gentamicin. Particular cultures were incubated with different concentrations (0.32 ng/ml, 0.8 ng/ml, 3.2 ng/ml, 8 ng/ml, 32 ng/ml) of recombinant human Epo (rhEpo, NeoRecormon, Roche, Welwyn Garden City, UK) or (0.42 ng/ml, 0.84 ng/ml) of the human non-erythropoietic Epo splice variant EV-3 (IBA GmbH, Göttingen, Germany) for another 3 days. Cell cultures were fixed in paraformaldehyde (4%, 30 min), labeled with DAPI (Sigma-Aldrich, Munich, Germany; 1:1000) and mounted with DABCO (Roth, Karlsruhe, Germany) on microscopic slides to quantify cellular survival (see below).
9
Quantifying Autophagy in Cell Lines
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HeLa or HEK293T eGFP-LC3B reporter cells were grown on coverslips (VWR) in 24-well plates and treated as indicated. THP-1 or Jurkat eGFP-LC3B reporter cells were grown in 24-well plates and treated as indicated. Cells were fixed with 4% (w/v) paraformaldehyde (PFA, Santa Cruz) for 20 min at RT, permeabilized with 0.5% (v/v) Triton-X-100 (Sigma-Aldrich) in PBS and then blocked with 5% (v/v) fetal bovine serum (Gibco) in PBS for 1 h at RT. Adherent cells were mounted on microscope slides (VWR) in 4′,6-diamidino-2-phenylindole (DAPI, VWR)-containing Mowiol mounting medium (Mowiol 4–88 10% (w/v, Carl Roth), glycerol 25% (w/v, Sigma-Aldrich), H2O 25% (v/v), 0.2 M Tris HCl pH 8.5 50% (v/v, AppliChem GmbH, VWR), DABCO 2.5% (w/v, Carl Roth)10 (link),38 (link)) to co-stain nuclei. The suspension cells were pelleted (300 g, 3 min), suspended in the same mounting medium, and mounted between coverslips and microscope slides (VWR). All laser scanning images were acquired on a Zeiss LSM 710 confocal microscope. The total area of cytoplasmic eGFP-LC3B puncta in HeLa, HEK293T, Jurkat and THP-1 cells was determined using a custom ImageJ macro for ≥ 30 randomly selected cells.
10
Immunohistochemistry of Hormone Receptors
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Glands were fixed with 4% paraformaldehyde overnight at 4 °C and embedded in paraffin. Sections measuring 4 μm were dewaxed, rehydrated and subjected to antigen retrieval with 10 mM trisodium citrate buffer, pH 6.0 for 20 min at 95 °C. Blocking of 1 h with 1% BSA was followed by incubation with primary and secondary antibodies. Primary antibodies were: rabbit anti-ERα (1:100–400; MC20, sc-542 SantaCruz), rabbit anti-PR (1:400; SP2, RM-9102 Thermo Fisher Scientific), anti-SMA (1:100; RB-9010-P0; Thermo Fisher Scientific), anti-p63 (1:100; MU418-UC; BioGenex) and rabbit anti-RFP (1:400; cat# PM005, MBL). All secondary antibodies were used at 1:500 dilutions (Molecular probes): alexa 488-conjugated anti-rabbit IgG, alexa 488-conjugated anti-goat IgG, alexa 568-conjugated anti-rabbit IgG and alexa 568-conjugated anti-mouse IgG. Nuclei were counterstained for 10 min with DAPI (Sigma) and mounted with Dabco (0718, Carl Roth). Images were acquired on confocal Zeiss LSM700 and reassembled with ImageJ software.
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