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Type 4 specificity titration. Type 1 anti-trastuzumab antibody AbD18018 in IgG1 format or trastuzumab were coated on a microtiter plate at a concentration of 5 µg/mL. After washing and blocking, the trastuzumab/Type 1 complex was formed by adding 5 µg/mL trastuzumab to the wells coated with the Type 1 antibody. Type 1 and trastuzumab negative controls were incubated with buffer only. After washing Type 4 antibody AbD23820 specific for the trastuzumab/Type 1 complex was titrated in <t>PBST</t> and added. Detection was performed using HRP-conjugated anti-Strep-tag antibody in HISPEC assay diluent followed by <t>QuantaBlu</t> fluorogenic peroxidase substrate.
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Images

1) Product Images from "Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics"

Article Title: Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics

Journal: mAbs

doi: 10.1080/19420862.2018.1538723

Type 4 specificity titration. Type 1 anti-trastuzumab antibody AbD18018 in IgG1 format or trastuzumab were coated on a microtiter plate at a concentration of 5 µg/mL. After washing and blocking, the trastuzumab/Type 1 complex was formed by adding 5 µg/mL trastuzumab to the wells coated with the Type 1 antibody. Type 1 and trastuzumab negative controls were incubated with buffer only. After washing Type 4 antibody AbD23820 specific for the trastuzumab/Type 1 complex was titrated in PBST and added. Detection was performed using HRP-conjugated anti-Strep-tag antibody in HISPEC assay diluent followed by QuantaBlu fluorogenic peroxidase substrate.
Figure Legend Snippet: Type 4 specificity titration. Type 1 anti-trastuzumab antibody AbD18018 in IgG1 format or trastuzumab were coated on a microtiter plate at a concentration of 5 µg/mL. After washing and blocking, the trastuzumab/Type 1 complex was formed by adding 5 µg/mL trastuzumab to the wells coated with the Type 1 antibody. Type 1 and trastuzumab negative controls were incubated with buffer only. After washing Type 4 antibody AbD23820 specific for the trastuzumab/Type 1 complex was titrated in PBST and added. Detection was performed using HRP-conjugated anti-Strep-tag antibody in HISPEC assay diluent followed by QuantaBlu fluorogenic peroxidase substrate.

Techniques Used: Titration, Concentration Assay, Blocking Assay, Incubation, Strep-tag

Demonstration of Type 3 antibody specificity. A microtiter plate was coated overnight with the omalizumab target human IgE, omalizumab, human IgG1/κ or human IgG1/λ at a concentration of 5 µg/mL. After washing and blocking with PBST + 5% BSA, the omalizumab/human IgE complex was formed by adding 2 µg/mL omalizumab to the wells coated with human IgE. Detection was performed with HRP-conjugated Type 3 anti-omalizumab/hIgE antibody AbD20760 in HISPEC assay diluent, followed by QuantaBlu fluorogenic peroxidase substrate.
Figure Legend Snippet: Demonstration of Type 3 antibody specificity. A microtiter plate was coated overnight with the omalizumab target human IgE, omalizumab, human IgG1/κ or human IgG1/λ at a concentration of 5 µg/mL. After washing and blocking with PBST + 5% BSA, the omalizumab/human IgE complex was formed by adding 2 µg/mL omalizumab to the wells coated with human IgE. Detection was performed with HRP-conjugated Type 3 anti-omalizumab/hIgE antibody AbD20760 in HISPEC assay diluent, followed by QuantaBlu fluorogenic peroxidase substrate.

Techniques Used: Concentration Assay, Blocking Assay

2) Product Images from "Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems"

Article Title: Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems

Journal: bioRxiv

doi: 10.1101/2020.05.13.094268

Demonstration of untargeted ExSeq with C. elegans (A), a Drosophila embryo (B) and the HeLa human cell lin e (C). The first in situ sequencing round is shown and the different colors (blue, magenta, green, and red) reveal the current base of the amplified cDNA (SOLiD sequencing was used). Scale bars: 20, 10 and 30 microns for panels A, B and C, respectively, in post-expansion (e.g., actual size) units. Methods used to generate this figure: Worm fixation and cuticle reduction was adopted from the published Bouin’s tube fixation protocol ( 127 ). The strain used in the figure was CZ1632: juIs76 [unc-25p::GFP + lin-15(+)] II. The strain was maintained at 20°C under standard conditions ( 128 ). The worms were collected from agar plates with M9 buffer (3g KH 2 PO 4 , 6g Na 2 HPO 4 , 5g NaCl, 1ml 1M MgSO 4 , water to 1 liter, sterilized by autoclaving) into a 15 mL tube. The tube was spun down at 1000g for 2 min, and the supernatant was replaced with 10 mL of fresh M9. The M9 wash step was repeated 2 more times. The worms were then transferred to a 1.5 mL tube and spun down to remove as much supernatant as possible without disturbing the worm pellet. The worms were placed on ice for 5 min. 1 mL of Bouin’s Fixative (0.46% picric acid, 4.4% paraformaldehyde, 2.4% acetic acid, 50% methanol, 1.2% 2-mercaptoethanol; as prepared in the published protocol), prepared fresh and pre-chilled to 4°C, was then added. The pellet was resuspended and mixed well. The sample was then placed on a tube rotator and mixed vigorously for 30 min at 25°C, followed by 4 hours of incubation at 4°C. The sample was then washed 3 times with 1mL Borate Triton β-mercaptoethanol solution (BTB; see recipe below); each time the sample was spun down, the supernatant was removed, the buffer was added and the sample was mixed thoroughly. BTB was prepared fresh using 1 mL 40x Borate Buffer Stock (3.1g boric acid, 1g NaOH, water to 50 mL), 1 mL 20% Triton X-100, 0.8 mL 2-mercaptoethanol, and 37.2 mL water. The sample was then further incubated three times, for 1 hour each, in 1 mL fresh BTB on a tube rotator at 25°C. Finally, the sample was washed six times: twice with 1 mL BT (1 mL 40x Borate Buffer Stock, 1 mL 20% Triton X-100, 38 mL water), twice with 1 mL 1x PBST (1x PBS, 0.5% Triton X-100), and twice with 1x PBS. The worms were permeabilized for 1 hour with 0.25% Triton X-100 in 1X PBS at 25°C. Drosophila larvae w1118 (BL#5905) were kindly provided by the lab of Aravinthan DT Samuel (Harvard University). Drosophila were raised in vials or bottles with standard yeast-containing medium at 22°C with alternating 12-h cycles of dark and light. HeLa (ATCC CCL-2) cells were cultured on CultureWell Chambered 16 wells Coverglass (Invitrogen) in D10 medium (Cellgro) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin–streptomycin (Cellgro), and 1% sodium pyruvate (BioWhittaker). Cultured cells were washed once with DPBS (Cellgro), fixed with 10% formalin in PBS for 15 min at 25°C, and washed three times with 1× PBS. Fixed cells were then stored in 70% ethanol at 4°C until use. ExSeq experimental procedures for the worms, Drosophila and HeLa cells were performed according to the following Methods sections: ‘RNA anchoring’, ‘Gelling, digestion and expansion’, ‘Re-embedding’, ‘Passivation’, and ‘Library preparation for in situ sequencing’. For the reverse transcription, instead of SSIV, M-MuLV (10U/µl; Enzymatics, cat. no. P7040L) was used for the worms and HeLa cells, whereas Maxima (10U/µl; Thermo Scientific, cat. no. EP0741) was used for Drosophila. Aminoallyl-dUTP was not included in the reverse transcription mix (and therefore the cDNA were not formalin-fixed), and the following primer sequence was used: /5Phos/ACTTCAGCTGCCCCGGGTGAAGANNNNNNNN. For the worms and HeLa cells, 2U/µl CircLigase II were used for circularization. To account for possible self-circularization of the primers, control samples with no reverse transcription enzyme were also processed for the worms, Drosophila, and HeLa cells, and as expected produced only a weak signal with hybridization probe after the library preparation. In situ sequencing was performed manually using the reagents and the enzymatic reactions outlined in the Methods section ‘Automated in situ sequencing’. Imaging was performed on a Zeiss Laser Scanning Confocal (LSM710) with Nikon 40X CFI Apo, water immersion with long working distance, NA 1.15 objective, and excitation light sources and emission filters as in ( 23 ).
Figure Legend Snippet: Demonstration of untargeted ExSeq with C. elegans (A), a Drosophila embryo (B) and the HeLa human cell lin e (C). The first in situ sequencing round is shown and the different colors (blue, magenta, green, and red) reveal the current base of the amplified cDNA (SOLiD sequencing was used). Scale bars: 20, 10 and 30 microns for panels A, B and C, respectively, in post-expansion (e.g., actual size) units. Methods used to generate this figure: Worm fixation and cuticle reduction was adopted from the published Bouin’s tube fixation protocol ( 127 ). The strain used in the figure was CZ1632: juIs76 [unc-25p::GFP + lin-15(+)] II. The strain was maintained at 20°C under standard conditions ( 128 ). The worms were collected from agar plates with M9 buffer (3g KH 2 PO 4 , 6g Na 2 HPO 4 , 5g NaCl, 1ml 1M MgSO 4 , water to 1 liter, sterilized by autoclaving) into a 15 mL tube. The tube was spun down at 1000g for 2 min, and the supernatant was replaced with 10 mL of fresh M9. The M9 wash step was repeated 2 more times. The worms were then transferred to a 1.5 mL tube and spun down to remove as much supernatant as possible without disturbing the worm pellet. The worms were placed on ice for 5 min. 1 mL of Bouin’s Fixative (0.46% picric acid, 4.4% paraformaldehyde, 2.4% acetic acid, 50% methanol, 1.2% 2-mercaptoethanol; as prepared in the published protocol), prepared fresh and pre-chilled to 4°C, was then added. The pellet was resuspended and mixed well. The sample was then placed on a tube rotator and mixed vigorously for 30 min at 25°C, followed by 4 hours of incubation at 4°C. The sample was then washed 3 times with 1mL Borate Triton β-mercaptoethanol solution (BTB; see recipe below); each time the sample was spun down, the supernatant was removed, the buffer was added and the sample was mixed thoroughly. BTB was prepared fresh using 1 mL 40x Borate Buffer Stock (3.1g boric acid, 1g NaOH, water to 50 mL), 1 mL 20% Triton X-100, 0.8 mL 2-mercaptoethanol, and 37.2 mL water. The sample was then further incubated three times, for 1 hour each, in 1 mL fresh BTB on a tube rotator at 25°C. Finally, the sample was washed six times: twice with 1 mL BT (1 mL 40x Borate Buffer Stock, 1 mL 20% Triton X-100, 38 mL water), twice with 1 mL 1x PBST (1x PBS, 0.5% Triton X-100), and twice with 1x PBS. The worms were permeabilized for 1 hour with 0.25% Triton X-100 in 1X PBS at 25°C. Drosophila larvae w1118 (BL#5905) were kindly provided by the lab of Aravinthan DT Samuel (Harvard University). Drosophila were raised in vials or bottles with standard yeast-containing medium at 22°C with alternating 12-h cycles of dark and light. HeLa (ATCC CCL-2) cells were cultured on CultureWell Chambered 16 wells Coverglass (Invitrogen) in D10 medium (Cellgro) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin–streptomycin (Cellgro), and 1% sodium pyruvate (BioWhittaker). Cultured cells were washed once with DPBS (Cellgro), fixed with 10% formalin in PBS for 15 min at 25°C, and washed three times with 1× PBS. Fixed cells were then stored in 70% ethanol at 4°C until use. ExSeq experimental procedures for the worms, Drosophila and HeLa cells were performed according to the following Methods sections: ‘RNA anchoring’, ‘Gelling, digestion and expansion’, ‘Re-embedding’, ‘Passivation’, and ‘Library preparation for in situ sequencing’. For the reverse transcription, instead of SSIV, M-MuLV (10U/µl; Enzymatics, cat. no. P7040L) was used for the worms and HeLa cells, whereas Maxima (10U/µl; Thermo Scientific, cat. no. EP0741) was used for Drosophila. Aminoallyl-dUTP was not included in the reverse transcription mix (and therefore the cDNA were not formalin-fixed), and the following primer sequence was used: /5Phos/ACTTCAGCTGCCCCGGGTGAAGANNNNNNNN. For the worms and HeLa cells, 2U/µl CircLigase II were used for circularization. To account for possible self-circularization of the primers, control samples with no reverse transcription enzyme were also processed for the worms, Drosophila, and HeLa cells, and as expected produced only a weak signal with hybridization probe after the library preparation. In situ sequencing was performed manually using the reagents and the enzymatic reactions outlined in the Methods section ‘Automated in situ sequencing’. Imaging was performed on a Zeiss Laser Scanning Confocal (LSM710) with Nikon 40X CFI Apo, water immersion with long working distance, NA 1.15 objective, and excitation light sources and emission filters as in ( 23 ).

Techniques Used: In Situ, Sequencing, Amplification, Incubation, Cell Culture, Produced, Hybridization, Imaging

3) Product Images from "In situ transduction of target cells on solid surfaces by immobilized viral vectors"

Article Title: In situ transduction of target cells on solid surfaces by immobilized viral vectors

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-3-4

Immobilization efficiency of biotinylated adenoviral vectors on streptavidin-coated wells. (A) Ad5.CMV-LacZ was treated with 15 μg/ml sulfo-NHS-LC-biotin, followed by the removal of non-virion-associated biotinylation reagent. Varying numbers of the resulting biotinylated adenoviral vectors (1 × 10 6 – 5 × 10 6 viral particles in 50 μl PBST per well) were incubated in streptavidin-coated wells (well diameter, 0.64 cm; Reacti-Bind Streptavidin Coated Polystyrene Wells) for 2 h at 25°C for immobilization. The solution of each well, which contained unbound viral particles, was collected and analyzed for the infectivity on D-17 cells (○). The same numbers of biotinylated Ad5.CMV-LacZ without application to streptavidin-coated wells were also analyzed for the infectivity on D-17 cells (●). The infectivity of immobilized viral particles was calculated by subtraction of the infectivity of unbound viral particles from the total infectivity of Ad5.CMV-LacZ applied to each well. The percentage of immobilized viral particles in the total is shown at the top. Each data point shown is the average + SD (n = 3). (B) Biotinylated Ad5.CMV-LacZ, prepared as above, was incubated in streptavidin-coated wells (2.5 × 10 6 viral particles per well) for 30 min at 25°C. The solution of each well was collected, and the wells were washed three times with PBST. Then, the same number of fresh biotinylated Ad5.CMV-LacZ was incubated in the same manner as above. These steps were repeated several more times. The solution in the well, which contained unbound viral particles, was collected after each addition cycle and titrated on D-17 cells (open bars). The Control bar shows the total infectivity of biotinylated Ad5.CMV-LacZ that was applied to wells at each addition cycle. The infectivity of immobilized viral particles (solid bars) was calculated by subtraction of the infectivity of unbound viral particles from the total infectivity of Ad5.CMV-LacZ applied at each addition cycle. The percentage of immobilized viral particles in the total at each addition cycle is shown at the top. Each data point shown is the average ± SD (n = 3).
Figure Legend Snippet: Immobilization efficiency of biotinylated adenoviral vectors on streptavidin-coated wells. (A) Ad5.CMV-LacZ was treated with 15 μg/ml sulfo-NHS-LC-biotin, followed by the removal of non-virion-associated biotinylation reagent. Varying numbers of the resulting biotinylated adenoviral vectors (1 × 10 6 – 5 × 10 6 viral particles in 50 μl PBST per well) were incubated in streptavidin-coated wells (well diameter, 0.64 cm; Reacti-Bind Streptavidin Coated Polystyrene Wells) for 2 h at 25°C for immobilization. The solution of each well, which contained unbound viral particles, was collected and analyzed for the infectivity on D-17 cells (○). The same numbers of biotinylated Ad5.CMV-LacZ without application to streptavidin-coated wells were also analyzed for the infectivity on D-17 cells (●). The infectivity of immobilized viral particles was calculated by subtraction of the infectivity of unbound viral particles from the total infectivity of Ad5.CMV-LacZ applied to each well. The percentage of immobilized viral particles in the total is shown at the top. Each data point shown is the average + SD (n = 3). (B) Biotinylated Ad5.CMV-LacZ, prepared as above, was incubated in streptavidin-coated wells (2.5 × 10 6 viral particles per well) for 30 min at 25°C. The solution of each well was collected, and the wells were washed three times with PBST. Then, the same number of fresh biotinylated Ad5.CMV-LacZ was incubated in the same manner as above. These steps were repeated several more times. The solution in the well, which contained unbound viral particles, was collected after each addition cycle and titrated on D-17 cells (open bars). The Control bar shows the total infectivity of biotinylated Ad5.CMV-LacZ that was applied to wells at each addition cycle. The infectivity of immobilized viral particles (solid bars) was calculated by subtraction of the infectivity of unbound viral particles from the total infectivity of Ad5.CMV-LacZ applied at each addition cycle. The percentage of immobilized viral particles in the total at each addition cycle is shown at the top. Each data point shown is the average ± SD (n = 3).

Techniques Used: Incubation, Infection

Regio-specific transduction of target cells on a solid surface by immobilized adenoviral vectors. Six-well cell culture plates, coated with poly- D -lysine (well diameter, 3.5 cm; Biocoat Poly- D -Lysine Cellware 6-Well Plates), were used as the solid surface. First, spots of biotin moieties on the wells were created by using sulfo-NHS-LC-biotin. Plastic rings (inner diameter, 1.4 cm) were used to specify biotinylation areas, and the remaining area of the wells was covered with a layer of 2% agarose. These spot areas within plastic rings were treated with 1 mg/ml sulfo-NHS-LC-biotin in PBS (pH 7.4) (50 μl per spot) for 2 h at 4°C, followed by the remove of unreacted biotinylation reagent. Neutralite avidin (25 μg in 50 μl PBST per spot) was added to the spot areas within the plastic rings and allowed to bind to biotin moieties of the well surface. Unbound Neutralite avidin was removed, and biotinylated Ad5.CMV-LacZ (1 × 10 7 viral particles per spot), prepared by treatment with 15 μg/ml sulfo-NHS-LC-biotin, was incubated in the spot areas within plastic rings at 25°C for 2 h, followed by the removal of unbound viral particles. The plastic rings and agarose were removed from the wells, which were then washed with PBS. D-17 cells (2.5 × 10 5 per well) were applied to the entirety of the wells, cultured at 37°C for 48 h, and stained for the expression of the lac Z gene.
Figure Legend Snippet: Regio-specific transduction of target cells on a solid surface by immobilized adenoviral vectors. Six-well cell culture plates, coated with poly- D -lysine (well diameter, 3.5 cm; Biocoat Poly- D -Lysine Cellware 6-Well Plates), were used as the solid surface. First, spots of biotin moieties on the wells were created by using sulfo-NHS-LC-biotin. Plastic rings (inner diameter, 1.4 cm) were used to specify biotinylation areas, and the remaining area of the wells was covered with a layer of 2% agarose. These spot areas within plastic rings were treated with 1 mg/ml sulfo-NHS-LC-biotin in PBS (pH 7.4) (50 μl per spot) for 2 h at 4°C, followed by the remove of unreacted biotinylation reagent. Neutralite avidin (25 μg in 50 μl PBST per spot) was added to the spot areas within the plastic rings and allowed to bind to biotin moieties of the well surface. Unbound Neutralite avidin was removed, and biotinylated Ad5.CMV-LacZ (1 × 10 7 viral particles per spot), prepared by treatment with 15 μg/ml sulfo-NHS-LC-biotin, was incubated in the spot areas within plastic rings at 25°C for 2 h, followed by the removal of unbound viral particles. The plastic rings and agarose were removed from the wells, which were then washed with PBS. D-17 cells (2.5 × 10 5 per well) were applied to the entirety of the wells, cultured at 37°C for 48 h, and stained for the expression of the lac Z gene.

Techniques Used: Transduction, Cell Culture, Avidin-Biotin Assay, Incubation, Staining, Expressing

4) Product Images from "Solid-Phase Capture of Proteins, Spores, and Bacteria †"

Article Title: Solid-Phase Capture of Proteins, Spores, and Bacteria †

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.67.3.1300-1307.2001

Standard curve of capture of B. globigii spores in PBST (A) and E. coli O157:H7 in meat extract and PBST (B) using 3-mm-diameter PEG-modified glass beads. Standard errors of the means are masked by the symbols. The y axis indicates absorbance.
Figure Legend Snippet: Standard curve of capture of B. globigii spores in PBST (A) and E. coli O157:H7 in meat extract and PBST (B) using 3-mm-diameter PEG-modified glass beads. Standard errors of the means are masked by the symbols. The y axis indicates absorbance.

Techniques Used: Modification

5) Product Images from "Solid-Phase Capture of Proteins, Spores, and Bacteria †"

Article Title: Solid-Phase Capture of Proteins, Spores, and Bacteria †

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.67.3.1300-1307.2001

Standard curve of capture of B. globigii spores in PBST (A) and E. coli O157:H7 in meat extract and PBST (B) using 3-mm-diameter PEG-modified glass beads. Standard errors of the means are masked by the symbols. The y axis indicates absorbance.
Figure Legend Snippet: Standard curve of capture of B. globigii spores in PBST (A) and E. coli O157:H7 in meat extract and PBST (B) using 3-mm-diameter PEG-modified glass beads. Standard errors of the means are masked by the symbols. The y axis indicates absorbance.

Techniques Used: Modification

6) Product Images from "Solid-Phase Capture of Proteins, Spores, and Bacteria †"

Article Title: Solid-Phase Capture of Proteins, Spores, and Bacteria †

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.67.3.1300-1307.2001

Standard curve of capture of B. globigii spores in PBST (A) and E. coli O157:H7 in meat extract and PBST (B) using 3-mm-diameter PEG-modified glass beads. Standard errors of the means are masked by the symbols. The y axis indicates absorbance.
Figure Legend Snippet: Standard curve of capture of B. globigii spores in PBST (A) and E. coli O157:H7 in meat extract and PBST (B) using 3-mm-diameter PEG-modified glass beads. Standard errors of the means are masked by the symbols. The y axis indicates absorbance.

Techniques Used: Modification

7) Product Images from "Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics"

Article Title: Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics

Journal: mAbs

doi: 10.1080/19420862.2018.1538723

Type 4 specificity titration. Type 1 anti-trastuzumab antibody AbD18018 in IgG1 format or trastuzumab were coated on a microtiter plate at a concentration of 5 µg/mL. After washing and blocking, the trastuzumab/Type 1 complex was formed by adding 5 µg/mL trastuzumab to the wells coated with the Type 1 antibody. Type 1 and trastuzumab negative controls were incubated with buffer only. After washing Type 4 antibody AbD23820 specific for the trastuzumab/Type 1 complex was titrated in PBST and added. Detection was performed using HRP-conjugated anti-Strep-tag antibody in HISPEC assay diluent followed by QuantaBlu fluorogenic peroxidase substrate.
Figure Legend Snippet: Type 4 specificity titration. Type 1 anti-trastuzumab antibody AbD18018 in IgG1 format or trastuzumab were coated on a microtiter plate at a concentration of 5 µg/mL. After washing and blocking, the trastuzumab/Type 1 complex was formed by adding 5 µg/mL trastuzumab to the wells coated with the Type 1 antibody. Type 1 and trastuzumab negative controls were incubated with buffer only. After washing Type 4 antibody AbD23820 specific for the trastuzumab/Type 1 complex was titrated in PBST and added. Detection was performed using HRP-conjugated anti-Strep-tag antibody in HISPEC assay diluent followed by QuantaBlu fluorogenic peroxidase substrate.

Techniques Used: Titration, Concentration Assay, Blocking Assay, Incubation, Strep-tag

Demonstration of Type 3 antibody specificity. A microtiter plate was coated overnight with the omalizumab target human IgE, omalizumab, human IgG1/κ or human IgG1/λ at a concentration of 5 µg/mL. After washing and blocking with PBST + 5% BSA, the omalizumab/human IgE complex was formed by adding 2 µg/mL omalizumab to the wells coated with human IgE. Detection was performed with HRP-conjugated Type 3 anti-omalizumab/hIgE antibody AbD20760 in HISPEC assay diluent, followed by QuantaBlu fluorogenic peroxidase substrate.
Figure Legend Snippet: Demonstration of Type 3 antibody specificity. A microtiter plate was coated overnight with the omalizumab target human IgE, omalizumab, human IgG1/κ or human IgG1/λ at a concentration of 5 µg/mL. After washing and blocking with PBST + 5% BSA, the omalizumab/human IgE complex was formed by adding 2 µg/mL omalizumab to the wells coated with human IgE. Detection was performed with HRP-conjugated Type 3 anti-omalizumab/hIgE antibody AbD20760 in HISPEC assay diluent, followed by QuantaBlu fluorogenic peroxidase substrate.

Techniques Used: Concentration Assay, Blocking Assay

8) Product Images from "Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems"

Article Title: Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems

Journal: bioRxiv

doi: 10.1101/2020.05.13.094268

Demonstration of untargeted ExSeq with C. elegans (A), a Drosophila embryo (B) and the HeLa human cell lin e (C). The first in situ sequencing round is shown and the different colors (blue, magenta, green, and red) reveal the current base of the amplified cDNA (SOLiD sequencing was used). Scale bars: 20, 10 and 30 microns for panels A, B and C, respectively, in post-expansion (e.g., actual size) units. Methods used to generate this figure: Worm fixation and cuticle reduction was adopted from the published Bouin’s tube fixation protocol ( 127 ). The strain used in the figure was CZ1632: juIs76 [unc-25p::GFP + lin-15(+)] II. The strain was maintained at 20°C under standard conditions ( 128 ). The worms were collected from agar plates with M9 buffer (3g KH 2 PO 4 , 6g Na 2 HPO 4 , 5g NaCl, 1ml 1M MgSO 4 , water to 1 liter, sterilized by autoclaving) into a 15 mL tube. The tube was spun down at 1000g for 2 min, and the supernatant was replaced with 10 mL of fresh M9. The M9 wash step was repeated 2 more times. The worms were then transferred to a 1.5 mL tube and spun down to remove as much supernatant as possible without disturbing the worm pellet. The worms were placed on ice for 5 min. 1 mL of Bouin’s Fixative (0.46% picric acid, 4.4% paraformaldehyde, 2.4% acetic acid, 50% methanol, 1.2% 2-mercaptoethanol; as prepared in the published protocol), prepared fresh and pre-chilled to 4°C, was then added. The pellet was resuspended and mixed well. The sample was then placed on a tube rotator and mixed vigorously for 30 min at 25°C, followed by 4 hours of incubation at 4°C. The sample was then washed 3 times with 1mL Borate Triton β-mercaptoethanol solution (BTB; see recipe below); each time the sample was spun down, the supernatant was removed, the buffer was added and the sample was mixed thoroughly. BTB was prepared fresh using 1 mL 40x Borate Buffer Stock (3.1g boric acid, 1g NaOH, water to 50 mL), 1 mL 20% Triton X-100, 0.8 mL 2-mercaptoethanol, and 37.2 mL water. The sample was then further incubated three times, for 1 hour each, in 1 mL fresh BTB on a tube rotator at 25°C. Finally, the sample was washed six times: twice with 1 mL BT (1 mL 40x Borate Buffer Stock, 1 mL 20% Triton X-100, 38 mL water), twice with 1 mL 1x PBST (1x PBS, 0.5% Triton X-100), and twice with 1x PBS. The worms were permeabilized for 1 hour with 0.25% Triton X-100 in 1X PBS at 25°C. Drosophila larvae w1118 (BL#5905) were kindly provided by the lab of Aravinthan DT Samuel (Harvard University). Drosophila were raised in vials or bottles with standard yeast-containing medium at 22°C with alternating 12-h cycles of dark and light. HeLa (ATCC CCL-2) cells were cultured on CultureWell Chambered 16 wells Coverglass (Invitrogen) in D10 medium (Cellgro) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin–streptomycin (Cellgro), and 1% sodium pyruvate (BioWhittaker). Cultured cells were washed once with DPBS (Cellgro), fixed with 10% formalin in PBS for 15 min at 25°C, and washed three times with 1× PBS. Fixed cells were then stored in 70% ethanol at 4°C until use. ExSeq experimental procedures for the worms, Drosophila and HeLa cells were performed according to the following Methods sections: ‘RNA anchoring’, ‘Gelling, digestion and expansion’, ‘Re-embedding’, ‘Passivation’, and ‘Library preparation for in situ sequencing’. For the reverse transcription, instead of SSIV, M-MuLV (10U/µl; Enzymatics, cat. no. P7040L) was used for the worms and HeLa cells, whereas Maxima (10U/µl; Thermo Scientific, cat. no. EP0741) was used for Drosophila. Aminoallyl-dUTP was not included in the reverse transcription mix (and therefore the cDNA were not formalin-fixed), and the following primer sequence was used: /5Phos/ACTTCAGCTGCCCCGGGTGAAGANNNNNNNN. For the worms and HeLa cells, 2U/µl CircLigase II were used for circularization. To account for possible self-circularization of the primers, control samples with no reverse transcription enzyme were also processed for the worms, Drosophila, and HeLa cells, and as expected produced only a weak signal with hybridization probe after the library preparation. In situ sequencing was performed manually using the reagents and the enzymatic reactions outlined in the Methods section ‘Automated in situ sequencing’. Imaging was performed on a Zeiss Laser Scanning Confocal (LSM710) with Nikon 40X CFI Apo, water immersion with long working distance, NA 1.15 objective, and excitation light sources and emission filters as in ( 23 ).
Figure Legend Snippet: Demonstration of untargeted ExSeq with C. elegans (A), a Drosophila embryo (B) and the HeLa human cell lin e (C). The first in situ sequencing round is shown and the different colors (blue, magenta, green, and red) reveal the current base of the amplified cDNA (SOLiD sequencing was used). Scale bars: 20, 10 and 30 microns for panels A, B and C, respectively, in post-expansion (e.g., actual size) units. Methods used to generate this figure: Worm fixation and cuticle reduction was adopted from the published Bouin’s tube fixation protocol ( 127 ). The strain used in the figure was CZ1632: juIs76 [unc-25p::GFP + lin-15(+)] II. The strain was maintained at 20°C under standard conditions ( 128 ). The worms were collected from agar plates with M9 buffer (3g KH 2 PO 4 , 6g Na 2 HPO 4 , 5g NaCl, 1ml 1M MgSO 4 , water to 1 liter, sterilized by autoclaving) into a 15 mL tube. The tube was spun down at 1000g for 2 min, and the supernatant was replaced with 10 mL of fresh M9. The M9 wash step was repeated 2 more times. The worms were then transferred to a 1.5 mL tube and spun down to remove as much supernatant as possible without disturbing the worm pellet. The worms were placed on ice for 5 min. 1 mL of Bouin’s Fixative (0.46% picric acid, 4.4% paraformaldehyde, 2.4% acetic acid, 50% methanol, 1.2% 2-mercaptoethanol; as prepared in the published protocol), prepared fresh and pre-chilled to 4°C, was then added. The pellet was resuspended and mixed well. The sample was then placed on a tube rotator and mixed vigorously for 30 min at 25°C, followed by 4 hours of incubation at 4°C. The sample was then washed 3 times with 1mL Borate Triton β-mercaptoethanol solution (BTB; see recipe below); each time the sample was spun down, the supernatant was removed, the buffer was added and the sample was mixed thoroughly. BTB was prepared fresh using 1 mL 40x Borate Buffer Stock (3.1g boric acid, 1g NaOH, water to 50 mL), 1 mL 20% Triton X-100, 0.8 mL 2-mercaptoethanol, and 37.2 mL water. The sample was then further incubated three times, for 1 hour each, in 1 mL fresh BTB on a tube rotator at 25°C. Finally, the sample was washed six times: twice with 1 mL BT (1 mL 40x Borate Buffer Stock, 1 mL 20% Triton X-100, 38 mL water), twice with 1 mL 1x PBST (1x PBS, 0.5% Triton X-100), and twice with 1x PBS. The worms were permeabilized for 1 hour with 0.25% Triton X-100 in 1X PBS at 25°C. Drosophila larvae w1118 (BL#5905) were kindly provided by the lab of Aravinthan DT Samuel (Harvard University). Drosophila were raised in vials or bottles with standard yeast-containing medium at 22°C with alternating 12-h cycles of dark and light. HeLa (ATCC CCL-2) cells were cultured on CultureWell Chambered 16 wells Coverglass (Invitrogen) in D10 medium (Cellgro) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin–streptomycin (Cellgro), and 1% sodium pyruvate (BioWhittaker). Cultured cells were washed once with DPBS (Cellgro), fixed with 10% formalin in PBS for 15 min at 25°C, and washed three times with 1× PBS. Fixed cells were then stored in 70% ethanol at 4°C until use. ExSeq experimental procedures for the worms, Drosophila and HeLa cells were performed according to the following Methods sections: ‘RNA anchoring’, ‘Gelling, digestion and expansion’, ‘Re-embedding’, ‘Passivation’, and ‘Library preparation for in situ sequencing’. For the reverse transcription, instead of SSIV, M-MuLV (10U/µl; Enzymatics, cat. no. P7040L) was used for the worms and HeLa cells, whereas Maxima (10U/µl; Thermo Scientific, cat. no. EP0741) was used for Drosophila. Aminoallyl-dUTP was not included in the reverse transcription mix (and therefore the cDNA were not formalin-fixed), and the following primer sequence was used: /5Phos/ACTTCAGCTGCCCCGGGTGAAGANNNNNNNN. For the worms and HeLa cells, 2U/µl CircLigase II were used for circularization. To account for possible self-circularization of the primers, control samples with no reverse transcription enzyme were also processed for the worms, Drosophila, and HeLa cells, and as expected produced only a weak signal with hybridization probe after the library preparation. In situ sequencing was performed manually using the reagents and the enzymatic reactions outlined in the Methods section ‘Automated in situ sequencing’. Imaging was performed on a Zeiss Laser Scanning Confocal (LSM710) with Nikon 40X CFI Apo, water immersion with long working distance, NA 1.15 objective, and excitation light sources and emission filters as in ( 23 ).

Techniques Used: In Situ, Sequencing, Amplification, Incubation, Cell Culture, Produced, Hybridization, Imaging

Related Articles

Concentration Assay:

Article Title: Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems
Article Snippet: .. These were labeled with a primary antibody against GFP (which labels the YFP protein) at a concentration of 10 µg/mL Rabbit Anti-GFP (Thermo Fisher, A-11122), in PBST overnight at 4°C followed by staining with a biotinylated secondary antibody at 10 µg/mL (Thermofisher, B-2770), in PBST overnight at 4°C. .. We note that antibody staining which is performed pre-expansion (as described in this section) results in a better staining compared to antibody staining which is performed post-sequencing (as described in the Methods section ‘Morphology’ above); however, in our hands, in situ sequencing with SOLiD chemistry was not successful for slices that were antibody stained pre-expansion.

Incubation:

Article Title: Solid-Phase Capture of Proteins, Spores, and Bacteria †
Article Snippet: .. Secondary Ab was added (total, 1012 molecules of anti- E. coli O157:H7, 1013 molecules of anti-OVA, 1013 molecules of anti-BSA, and 1012 molecules of anti- B. globigii ) in 10 ml of PBST, and beads were again incubated for 1 h. Samples were washed six times with 50 ml of PBST (pH 5.8) and incubated with 10 ml of anti-IgG conjugated to HRP (Pierce) (IgG-HRP, 1 μg per 10 ml of PBST [pH 5.8]). .. After the last wash step, beads were added to 5 ml of 1-Step Turbo TMB-ELISA substrate (Pierce) and incubated in the dark for 20 min before an A 370 reading was taken using a Cary 100-Bio UV/visible light spectrophotometer (Varian, Sugar Land, Tex.).

Article Title: In situ transduction of target cells on solid surfaces by immobilized viral vectors
Article Snippet: .. Preparation of wells coated with adenoviral vectors Biotinylated Ad5.CMV-LacZ, prepared by treatment with 15 μg/ml sulfo-NHS-LC-biotin as above, was diluted in PBST and incubated for immobilization in streptavidin-coated wells (well diameter, 0.64 cm; Reacti-Bind Streptavidin Coated Polystyrene Wells, Pierce Chemical) (50 μl per well) for 2 h at 25°C with shaking on a rotary shaker at 150 rpm. .. The solution of each well, which contained unbound Ad5.CMV-LacZ, was collected and analyzed for infectivity on D-17 cells as above to estimate the number of unbound viral particles.

Labeling:

Article Title: Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems
Article Snippet: .. These were labeled with a primary antibody against GFP (which labels the YFP protein) at a concentration of 10 µg/mL Rabbit Anti-GFP (Thermo Fisher, A-11122), in PBST overnight at 4°C followed by staining with a biotinylated secondary antibody at 10 µg/mL (Thermofisher, B-2770), in PBST overnight at 4°C. .. We note that antibody staining which is performed pre-expansion (as described in this section) results in a better staining compared to antibody staining which is performed post-sequencing (as described in the Methods section ‘Morphology’ above); however, in our hands, in situ sequencing with SOLiD chemistry was not successful for slices that were antibody stained pre-expansion.

Staining:

Article Title: Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems
Article Snippet: .. These were labeled with a primary antibody against GFP (which labels the YFP protein) at a concentration of 10 µg/mL Rabbit Anti-GFP (Thermo Fisher, A-11122), in PBST overnight at 4°C followed by staining with a biotinylated secondary antibody at 10 µg/mL (Thermofisher, B-2770), in PBST overnight at 4°C. .. We note that antibody staining which is performed pre-expansion (as described in this section) results in a better staining compared to antibody staining which is performed post-sequencing (as described in the Methods section ‘Morphology’ above); however, in our hands, in situ sequencing with SOLiD chemistry was not successful for slices that were antibody stained pre-expansion.

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    Type 4 specificity titration. Type 1 anti-trastuzumab antibody AbD18018 in IgG1 format or trastuzumab were coated on a microtiter plate at a concentration of 5 µg/mL. After washing and blocking, the trastuzumab/Type 1 complex was formed by adding 5 µg/mL trastuzumab to the wells coated with the Type 1 antibody. Type 1 and trastuzumab negative controls were incubated with buffer only. After washing Type 4 antibody AbD23820 specific for the trastuzumab/Type 1 complex was titrated in <t>PBST</t> and added. Detection was performed using HRP-conjugated anti-Strep-tag antibody in HISPEC assay diluent followed by <t>QuantaBlu</t> fluorogenic peroxidase substrate.
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    Type 4 specificity titration. Type 1 anti-trastuzumab antibody AbD18018 in IgG1 format or trastuzumab were coated on a microtiter plate at a concentration of 5 µg/mL. After washing and blocking, the trastuzumab/Type 1 complex was formed by adding 5 µg/mL trastuzumab to the wells coated with the Type 1 antibody. Type 1 and trastuzumab negative controls were incubated with buffer only. After washing Type 4 antibody AbD23820 specific for the trastuzumab/Type 1 complex was titrated in PBST and added. Detection was performed using HRP-conjugated anti-Strep-tag antibody in HISPEC assay diluent followed by QuantaBlu fluorogenic peroxidase substrate.

    Journal: mAbs

    Article Title: Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics

    doi: 10.1080/19420862.2018.1538723

    Figure Lengend Snippet: Type 4 specificity titration. Type 1 anti-trastuzumab antibody AbD18018 in IgG1 format or trastuzumab were coated on a microtiter plate at a concentration of 5 µg/mL. After washing and blocking, the trastuzumab/Type 1 complex was formed by adding 5 µg/mL trastuzumab to the wells coated with the Type 1 antibody. Type 1 and trastuzumab negative controls were incubated with buffer only. After washing Type 4 antibody AbD23820 specific for the trastuzumab/Type 1 complex was titrated in PBST and added. Detection was performed using HRP-conjugated anti-Strep-tag antibody in HISPEC assay diluent followed by QuantaBlu fluorogenic peroxidase substrate.

    Article Snippet: Plates were washed ten times with PBST, followed by the addition of QuantaBlu fluorogenic peroxidase substrate (Thermo Fisher Scientific #15169).

    Techniques: Titration, Concentration Assay, Blocking Assay, Incubation, Strep-tag

    Demonstration of Type 3 antibody specificity. A microtiter plate was coated overnight with the omalizumab target human IgE, omalizumab, human IgG1/κ or human IgG1/λ at a concentration of 5 µg/mL. After washing and blocking with PBST + 5% BSA, the omalizumab/human IgE complex was formed by adding 2 µg/mL omalizumab to the wells coated with human IgE. Detection was performed with HRP-conjugated Type 3 anti-omalizumab/hIgE antibody AbD20760 in HISPEC assay diluent, followed by QuantaBlu fluorogenic peroxidase substrate.

    Journal: mAbs

    Article Title: Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics

    doi: 10.1080/19420862.2018.1538723

    Figure Lengend Snippet: Demonstration of Type 3 antibody specificity. A microtiter plate was coated overnight with the omalizumab target human IgE, omalizumab, human IgG1/κ or human IgG1/λ at a concentration of 5 µg/mL. After washing and blocking with PBST + 5% BSA, the omalizumab/human IgE complex was formed by adding 2 µg/mL omalizumab to the wells coated with human IgE. Detection was performed with HRP-conjugated Type 3 anti-omalizumab/hIgE antibody AbD20760 in HISPEC assay diluent, followed by QuantaBlu fluorogenic peroxidase substrate.

    Article Snippet: Plates were washed ten times with PBST, followed by the addition of QuantaBlu fluorogenic peroxidase substrate (Thermo Fisher Scientific #15169).

    Techniques: Concentration Assay, Blocking Assay

    Demonstration of untargeted ExSeq with C. elegans (A), a Drosophila embryo (B) and the HeLa human cell lin e (C). The first in situ sequencing round is shown and the different colors (blue, magenta, green, and red) reveal the current base of the amplified cDNA (SOLiD sequencing was used). Scale bars: 20, 10 and 30 microns for panels A, B and C, respectively, in post-expansion (e.g., actual size) units. Methods used to generate this figure: Worm fixation and cuticle reduction was adopted from the published Bouin’s tube fixation protocol ( 127 ). The strain used in the figure was CZ1632: juIs76 [unc-25p::GFP + lin-15(+)] II. The strain was maintained at 20°C under standard conditions ( 128 ). The worms were collected from agar plates with M9 buffer (3g KH 2 PO 4 , 6g Na 2 HPO 4 , 5g NaCl, 1ml 1M MgSO 4 , water to 1 liter, sterilized by autoclaving) into a 15 mL tube. The tube was spun down at 1000g for 2 min, and the supernatant was replaced with 10 mL of fresh M9. The M9 wash step was repeated 2 more times. The worms were then transferred to a 1.5 mL tube and spun down to remove as much supernatant as possible without disturbing the worm pellet. The worms were placed on ice for 5 min. 1 mL of Bouin’s Fixative (0.46% picric acid, 4.4% paraformaldehyde, 2.4% acetic acid, 50% methanol, 1.2% 2-mercaptoethanol; as prepared in the published protocol), prepared fresh and pre-chilled to 4°C, was then added. The pellet was resuspended and mixed well. The sample was then placed on a tube rotator and mixed vigorously for 30 min at 25°C, followed by 4 hours of incubation at 4°C. The sample was then washed 3 times with 1mL Borate Triton β-mercaptoethanol solution (BTB; see recipe below); each time the sample was spun down, the supernatant was removed, the buffer was added and the sample was mixed thoroughly. BTB was prepared fresh using 1 mL 40x Borate Buffer Stock (3.1g boric acid, 1g NaOH, water to 50 mL), 1 mL 20% Triton X-100, 0.8 mL 2-mercaptoethanol, and 37.2 mL water. The sample was then further incubated three times, for 1 hour each, in 1 mL fresh BTB on a tube rotator at 25°C. Finally, the sample was washed six times: twice with 1 mL BT (1 mL 40x Borate Buffer Stock, 1 mL 20% Triton X-100, 38 mL water), twice with 1 mL 1x PBST (1x PBS, 0.5% Triton X-100), and twice with 1x PBS. The worms were permeabilized for 1 hour with 0.25% Triton X-100 in 1X PBS at 25°C. Drosophila larvae w1118 (BL#5905) were kindly provided by the lab of Aravinthan DT Samuel (Harvard University). Drosophila were raised in vials or bottles with standard yeast-containing medium at 22°C with alternating 12-h cycles of dark and light. HeLa (ATCC CCL-2) cells were cultured on CultureWell Chambered 16 wells Coverglass (Invitrogen) in D10 medium (Cellgro) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin–streptomycin (Cellgro), and 1% sodium pyruvate (BioWhittaker). Cultured cells were washed once with DPBS (Cellgro), fixed with 10% formalin in PBS for 15 min at 25°C, and washed three times with 1× PBS. Fixed cells were then stored in 70% ethanol at 4°C until use. ExSeq experimental procedures for the worms, Drosophila and HeLa cells were performed according to the following Methods sections: ‘RNA anchoring’, ‘Gelling, digestion and expansion’, ‘Re-embedding’, ‘Passivation’, and ‘Library preparation for in situ sequencing’. For the reverse transcription, instead of SSIV, M-MuLV (10U/µl; Enzymatics, cat. no. P7040L) was used for the worms and HeLa cells, whereas Maxima (10U/µl; Thermo Scientific, cat. no. EP0741) was used for Drosophila. Aminoallyl-dUTP was not included in the reverse transcription mix (and therefore the cDNA were not formalin-fixed), and the following primer sequence was used: /5Phos/ACTTCAGCTGCCCCGGGTGAAGANNNNNNNN. For the worms and HeLa cells, 2U/µl CircLigase II were used for circularization. To account for possible self-circularization of the primers, control samples with no reverse transcription enzyme were also processed for the worms, Drosophila, and HeLa cells, and as expected produced only a weak signal with hybridization probe after the library preparation. In situ sequencing was performed manually using the reagents and the enzymatic reactions outlined in the Methods section ‘Automated in situ sequencing’. Imaging was performed on a Zeiss Laser Scanning Confocal (LSM710) with Nikon 40X CFI Apo, water immersion with long working distance, NA 1.15 objective, and excitation light sources and emission filters as in ( 23 ).

    Journal: bioRxiv

    Article Title: Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems

    doi: 10.1101/2020.05.13.094268

    Figure Lengend Snippet: Demonstration of untargeted ExSeq with C. elegans (A), a Drosophila embryo (B) and the HeLa human cell lin e (C). The first in situ sequencing round is shown and the different colors (blue, magenta, green, and red) reveal the current base of the amplified cDNA (SOLiD sequencing was used). Scale bars: 20, 10 and 30 microns for panels A, B and C, respectively, in post-expansion (e.g., actual size) units. Methods used to generate this figure: Worm fixation and cuticle reduction was adopted from the published Bouin’s tube fixation protocol ( 127 ). The strain used in the figure was CZ1632: juIs76 [unc-25p::GFP + lin-15(+)] II. The strain was maintained at 20°C under standard conditions ( 128 ). The worms were collected from agar plates with M9 buffer (3g KH 2 PO 4 , 6g Na 2 HPO 4 , 5g NaCl, 1ml 1M MgSO 4 , water to 1 liter, sterilized by autoclaving) into a 15 mL tube. The tube was spun down at 1000g for 2 min, and the supernatant was replaced with 10 mL of fresh M9. The M9 wash step was repeated 2 more times. The worms were then transferred to a 1.5 mL tube and spun down to remove as much supernatant as possible without disturbing the worm pellet. The worms were placed on ice for 5 min. 1 mL of Bouin’s Fixative (0.46% picric acid, 4.4% paraformaldehyde, 2.4% acetic acid, 50% methanol, 1.2% 2-mercaptoethanol; as prepared in the published protocol), prepared fresh and pre-chilled to 4°C, was then added. The pellet was resuspended and mixed well. The sample was then placed on a tube rotator and mixed vigorously for 30 min at 25°C, followed by 4 hours of incubation at 4°C. The sample was then washed 3 times with 1mL Borate Triton β-mercaptoethanol solution (BTB; see recipe below); each time the sample was spun down, the supernatant was removed, the buffer was added and the sample was mixed thoroughly. BTB was prepared fresh using 1 mL 40x Borate Buffer Stock (3.1g boric acid, 1g NaOH, water to 50 mL), 1 mL 20% Triton X-100, 0.8 mL 2-mercaptoethanol, and 37.2 mL water. The sample was then further incubated three times, for 1 hour each, in 1 mL fresh BTB on a tube rotator at 25°C. Finally, the sample was washed six times: twice with 1 mL BT (1 mL 40x Borate Buffer Stock, 1 mL 20% Triton X-100, 38 mL water), twice with 1 mL 1x PBST (1x PBS, 0.5% Triton X-100), and twice with 1x PBS. The worms were permeabilized for 1 hour with 0.25% Triton X-100 in 1X PBS at 25°C. Drosophila larvae w1118 (BL#5905) were kindly provided by the lab of Aravinthan DT Samuel (Harvard University). Drosophila were raised in vials or bottles with standard yeast-containing medium at 22°C with alternating 12-h cycles of dark and light. HeLa (ATCC CCL-2) cells were cultured on CultureWell Chambered 16 wells Coverglass (Invitrogen) in D10 medium (Cellgro) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin–streptomycin (Cellgro), and 1% sodium pyruvate (BioWhittaker). Cultured cells were washed once with DPBS (Cellgro), fixed with 10% formalin in PBS for 15 min at 25°C, and washed three times with 1× PBS. Fixed cells were then stored in 70% ethanol at 4°C until use. ExSeq experimental procedures for the worms, Drosophila and HeLa cells were performed according to the following Methods sections: ‘RNA anchoring’, ‘Gelling, digestion and expansion’, ‘Re-embedding’, ‘Passivation’, and ‘Library preparation for in situ sequencing’. For the reverse transcription, instead of SSIV, M-MuLV (10U/µl; Enzymatics, cat. no. P7040L) was used for the worms and HeLa cells, whereas Maxima (10U/µl; Thermo Scientific, cat. no. EP0741) was used for Drosophila. Aminoallyl-dUTP was not included in the reverse transcription mix (and therefore the cDNA were not formalin-fixed), and the following primer sequence was used: /5Phos/ACTTCAGCTGCCCCGGGTGAAGANNNNNNNN. For the worms and HeLa cells, 2U/µl CircLigase II were used for circularization. To account for possible self-circularization of the primers, control samples with no reverse transcription enzyme were also processed for the worms, Drosophila, and HeLa cells, and as expected produced only a weak signal with hybridization probe after the library preparation. In situ sequencing was performed manually using the reagents and the enzymatic reactions outlined in the Methods section ‘Automated in situ sequencing’. Imaging was performed on a Zeiss Laser Scanning Confocal (LSM710) with Nikon 40X CFI Apo, water immersion with long working distance, NA 1.15 objective, and excitation light sources and emission filters as in ( 23 ).

    Article Snippet: These were labeled with a primary antibody against GFP (which labels the YFP protein) at a concentration of 10 µg/mL Rabbit Anti-GFP (Thermo Fisher, A-11122), in PBST overnight at 4°C followed by staining with a biotinylated secondary antibody at 10 µg/mL (Thermofisher, B-2770), in PBST overnight at 4°C.

    Techniques: In Situ, Sequencing, Amplification, Incubation, Cell Culture, Produced, Hybridization, Imaging

    Type 4 specificity titration. Type 1 anti-trastuzumab antibody AbD18018 in IgG1 format or trastuzumab were coated on a microtiter plate at a concentration of 5 µg/mL. After washing and blocking, the trastuzumab/Type 1 complex was formed by adding 5 µg/mL trastuzumab to the wells coated with the Type 1 antibody. Type 1 and trastuzumab negative controls were incubated with buffer only. After washing Type 4 antibody AbD23820 specific for the trastuzumab/Type 1 complex was titrated in PBST and added. Detection was performed using HRP-conjugated anti-Strep-tag antibody in HISPEC assay diluent followed by QuantaBlu fluorogenic peroxidase substrate.

    Journal: mAbs

    Article Title: Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics

    doi: 10.1080/19420862.2018.1538723

    Figure Lengend Snippet: Type 4 specificity titration. Type 1 anti-trastuzumab antibody AbD18018 in IgG1 format or trastuzumab were coated on a microtiter plate at a concentration of 5 µg/mL. After washing and blocking, the trastuzumab/Type 1 complex was formed by adding 5 µg/mL trastuzumab to the wells coated with the Type 1 antibody. Type 1 and trastuzumab negative controls were incubated with buffer only. After washing Type 4 antibody AbD23820 specific for the trastuzumab/Type 1 complex was titrated in PBST and added. Detection was performed using HRP-conjugated anti-Strep-tag antibody in HISPEC assay diluent followed by QuantaBlu fluorogenic peroxidase substrate.

    Article Snippet: Plates were washed ten times with PBST, followed by the addition of QuantaBlu fluorogenic peroxidase substrate (Thermo Fisher Scientific #15169).

    Techniques: Titration, Concentration Assay, Blocking Assay, Incubation, Strep-tag

    Demonstration of Type 3 antibody specificity. A microtiter plate was coated overnight with the omalizumab target human IgE, omalizumab, human IgG1/κ or human IgG1/λ at a concentration of 5 µg/mL. After washing and blocking with PBST + 5% BSA, the omalizumab/human IgE complex was formed by adding 2 µg/mL omalizumab to the wells coated with human IgE. Detection was performed with HRP-conjugated Type 3 anti-omalizumab/hIgE antibody AbD20760 in HISPEC assay diluent, followed by QuantaBlu fluorogenic peroxidase substrate.

    Journal: mAbs

    Article Title: Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics

    doi: 10.1080/19420862.2018.1538723

    Figure Lengend Snippet: Demonstration of Type 3 antibody specificity. A microtiter plate was coated overnight with the omalizumab target human IgE, omalizumab, human IgG1/κ or human IgG1/λ at a concentration of 5 µg/mL. After washing and blocking with PBST + 5% BSA, the omalizumab/human IgE complex was formed by adding 2 µg/mL omalizumab to the wells coated with human IgE. Detection was performed with HRP-conjugated Type 3 anti-omalizumab/hIgE antibody AbD20760 in HISPEC assay diluent, followed by QuantaBlu fluorogenic peroxidase substrate.

    Article Snippet: Plates were washed ten times with PBST, followed by the addition of QuantaBlu fluorogenic peroxidase substrate (Thermo Fisher Scientific #15169).

    Techniques: Concentration Assay, Blocking Assay