Flies were cultivated and crossed in vials containing potato mash-yeast-agar. We used the Oregon R strain as wild type, and w; P[w+hsp83:msl2] msl3/ TM6B females (from stock kindly provided by Mitzi Kuroda) to express MSL2 in an msl3 mutant background in order to visualize high-affinity sites staining on polytene chromosomes (Dahlsveen et al. 2006 (link); Demakova et al. 2003 (link); Kelley et al. 1995 (link)) and compare their patterns to PLA staining patterns. Polytene chromosomes from the salivary glands of the third instar larvae were prepared essentially as previously described (Johansson et al. 2012 (link); Lundberg et al. 2013 ). Briefly, salivary glands were dissected and fixed in 3.7 % formaldehyde in PBS, 0.3 % Triton X-100, for 40 s, followed by 2–3 min in 50 % acetic acid containing 1 % formaldehyde. Polytene chromosomes were squashed with high pressure using a MTC-200-1 precision vice (Penn Tool: Maplewood, NJ) as previously described by Novikov et al. (2007 (link)). The slides were quick-frozen in liquid nitrogen; the coverslip was removed; and the slides were stored in ethanol at −20 °C until required for use. Just before antibody incubation or in situ hybridization, the slides were air-dried and the areas with chromosome spreads were encircled using an ImmEdge Pen (Vector Laboratories).
Immedge pen
Manufactured by Vector Laboratories
Sourced in Canada, United States
The ImmEdge pen is a laboratory tool used for applying hydrophobic barriers. It features a fine-tipped applicator that allows for precise and controlled application of the barrier material.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (6 mentions)
Normal goat donkey serum, by Vector Laboratories (3 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (3 mentions)
Normal goat serum (ngs), by Vector Laboratories (2 mentions)
Ultrapure bsa, by Thermo Fisher Scientific (2 mentions)
Tris buffered saline (tbs), by Biocare Medical (2 mentions)
Cytoclear glass slide, by Sterlitech (2 mentions)
Vectorshield mounting medium, by Vector Laboratories (2 mentions)
Rnascope fluorescent multiplex kit, by Advanced Cell Diagnostics (2 mentions)
Hoechst nuclear stain, by Merck Group (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Heparinase 2, by Merck Group (1 mentions)
Vectorshield mounting medium containing dapi, by Vector Laboratories (1 mentions)
Fluorophore conjugated secondary antibodies, by Cell Signaling Technology (1 mentions)
Cytoseal xyl, by Thermo Fisher Scientific (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
Rnascope multiplex fluorescent reagent kit v2, by Advanced Cell Diagnostics (1 mentions)
Quantigene viewrna ish tissue assay, by Thermo Fisher Scientific (1 mentions)
Histoclear, by National Diagnostics (1 mentions)
37 protocols using immedge pen
1
Visualizing High-Affinity Sites on Polytene Chromosomes
2
Cathepsin B and Heparinase II Protocol
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Frozen lymph node or tonsil sections on polylysine slides were incubated at room temperature for 30 min. A circle was drawn around each section using a wax ImmEdge pen (Vector Laboratories), the sections were then hydrated with PBS for 5 min and incubated with 150 nM recombinant Cath-B (Sigma-Aldrich) for 3 h at 37 °C or with 10 U heparinase II (Sigma-Aldrich) for 1 h at 17 °C. Slides were washed in PBS and then processed for immunohistochemistry (as described below) with no fixative. All samples were ethically approved and informed consent was obtained from all participants. Tonsils were collected under NRES REC 12/NE/0360-approved study (IRAS: 114771) to M.C.C. Hepatic lymph nodes were collected during multiorgan donation procedures, after approval by the Medical Ethical committee of the Erasmus MC (MEC-2014-060) by WGP.
3
Immunohistochemistry on Tissue Sections
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Mounted tissue sections were equilibrated to room temperature, hydrated in PBS for 2 × 5 minutes, permeabilized in 0.1% Triton X‐100 in PBS for 20 minutes at room temperature and washed for 2 × 5 minutes in PBS before isolation with a hydrophobic PAP pen (Immedge pen, Vector Laboratories). Nonspecific protein binding sites in sections were blocked by incubation in blocking buffer (75 µl; 0.5% bovine serum albumin (g/ml), 0.3% Tween 20, 15% normal goat/donkey serum (Vector Laboratories) in PBS) in a humidified chamber for 30 minutes at room temperature and then sections were drained and incubated with primary antibody (Supporting Information Table 1) diluted in ADB (15% normal goat serum in place of bovine serum albumin) overnight at 4°C. The following day, slides were washed for 3 × 5 minutes in PBS. Tissue sections were then incubated with secondary antibody diluted in ADB for 1 hour in a hydrated incubation chamber at room temperature. After 1 hour, slides were washed for 3 × 5 minutes in PBS, mounted in Vectorshield mounting medium containing DAPI (Vector Laboratories) and stored at 4°C before microscopic analysis.
4
Immunofluorescence Staining of Tissue Sections
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Human or mouse tissue sections were marked on the edge with ImmEdge pen (H-4000, Vector Labs) and fixed in 4% Paraformaldehyde (PFA) for 15 min. Human and mouse tissues were blocked with 5% human or mouse serum, respectively, for 1 hour at room temperature (RT). This was followed by incubation with fluorophore-conjugated (1:100 dilution) or non-conjugated primary antibodies (1/500 dilution) for 3 hours at RT. Slides were washed four times in 1× PBS. To visualize antigens targeted with non-conjugated primary antibodies, fluorophore-conjugated secondary antibodies (1:1000 dilution, Cell Signaling Technology) were added along with nuclear dye (Hoechst 33422, #H1399; Thermo Fisher Scientific) for 30 min at RT, washed 4 four times in 1× PBS, and sections were mounted with coverslips in prolonged gold mounting medium. Tissues were analyzed in Leica SP8 DMRE spectral confocal microscope, and Image J software was used for postimage analysis. The full details of antibodies and secondary antibodies used for confocal staining of human and mouse tissues are given in online supplemental table 2 ).
5
Immunofluorescence Staining for Protein Localization
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Slides were washed in 1% Triton X-100 in RNase-free 1X PBS and rocked gently for 10 min. Slides were washed twice for 5 min in RNase-free 1X PBS the sample perimeter of each slide was marked with an ImmEdge pen (Vector Laboratories #H-4000) in between both washes. 250 µL of blocking solution (0.5% UltraPure BSA (Invitrogen #AM2616) in RNase-free 1X PBS) was added to the sample area of each slide and the slides were incubated at room temperature in a dark humid chamber for 1 h, shaking gently. Using coverslips, slides were incubated with 40 µL of diluted primary antibody in blocking solution overnight in a dark, sealed, humid chamber at 4°C. Slides were washed 3 times for 5 min in RNase-free 1X PBS. 40ul of diluted secondary antibody in blocking solution was applied to each slide (including coverslips) and slides were incubated for 2 h in a dark humid chamber at room temperature. Slides were washed 3 times for 5 min in RNase-free 1X PBS. 250 µL of post-fixative (4% paraformaldehyde in RNase-free 1X PBS) was added to each sample area for 3 min at room temperature. Each slide was then washed 3 times for 5 min in RNase-free 1X PBS.
6
Immunohistochemical Analysis of Lung Tissues
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Control lung tissues were obtained from otherwise healthy, non-smoking patients undergoing surgery for lung cancer (four donors) while CF lung tissues were obtained from patients suffering from CF, undergoing lung transplantation (four donors) (Supplemental Table I
7
Urine Filtration for Cell Analysis
8
Click Chemistry Biotin Labeling
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An ImmEdge™ Pen (Vector Laboratories, H-4000) was used for drawing a water-repellent barrier around the tissue section that was mounted on a microscope slide. The tissue specimen was washed with PBS twice, fixed with 4% PFA for 10 min, then quenched with 50 mM NH4Cl/glycine in PBS. The click conversion of N3 labelling was performed with 10 μM biotin-PEG4-alkyne, 100 μM CuSO4, 200 μM BTTAA and 2.5 mM sodium ascorbate in PBS at room temperature for 30 min. The tissue specimen was washed with PBS five times after click reaction.
9
Immunohistochemical Analysis Protocol
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For immunohistochemical analysis, 4-μm sections were deparaffinized with xylene three times, washed with 100% ethanol to remove xylene, and rehydrated in a graded series of ethanol. Slides were placed in a boiling water bath for 20 min and then cooled at room temperature for 30 min. Slides were washed with water and treated with 0.3% hydrogen peroxide in methanol for 10 min. Excess moisture was removed, and tissues were encircled by ImmEdge pen (Vector Laboratories, Burlingame, CA, USA). Slides were washed in PBS and then blocked for 1 h with blocking buffer (PBS + 1% BSA + 20% horse serum). Block solution was aspirated away and primary antibody (4 μg/ml) applied in a humidity chamber at 4°C overnight. After a PBS wash, secondary mouse antibody (1:100 diluted, Vector Laboratories) was incubated on slides for 1 h at room temperature, followed by PBS wash. ABC Standard Elite kit (Vector Laboratories, PK-6100) solution was applied to the slides for 30 min at room temperature and washed away with PBS. DAB reagent was applied to develop the slides, which were then washed with PBS, counterstained with standard H&E, and mounted with Cytoseal XYL (Richard-Allan Scientific, Kalamazoo, MI, USA).
10
Immunohistochemical Analysis of Uterine Receptors
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Likewise, the paraffin-embedded uteri were cross-sectioned at 4 μm,
deparaffinized and hydrated. Then, they were subjected to antigen retrieval in
boiling 10 mM sodium citrate buffer (pH 6.0) for 10 min. The tissues were
permeabilized with PBST for 5 min. Hydrophobic barriers were drawn surrounding
the tissues with ImmEdge™ Pen (Vector Laboratories, Inc., Burlingame,
CA). For blocking, the tissues were incubated with 1% normal blocking
serum in PBS for 1 h, and then incubated with antibody of each ESR1, ESR2, or
PGR for 1 h (Table 1 ). After washed in
PBST and PBS, for PGR detection tissues were incubated with second antibodies
for 1 h and washed, but for ESR1 or ESR2 this step was skipped because the first
antibodies were fluorescence conjugated. The tissues were counterstained with
YOYO-1 (Cat #: Y3601, Invitrogen, Waltham, MA) for 15 min and mounted.
Specific signaling of ESR1, ESR2, or PGR was observed under the fluorescence
microscope (Zeiss Axio Observer Z1).
deparaffinized and hydrated. Then, they were subjected to antigen retrieval in
boiling 10 mM sodium citrate buffer (pH 6.0) for 10 min. The tissues were
permeabilized with PBST for 5 min. Hydrophobic barriers were drawn surrounding
the tissues with ImmEdge™ Pen (Vector Laboratories, Inc., Burlingame,
CA). For blocking, the tissues were incubated with 1% normal blocking
serum in PBS for 1 h, and then incubated with antibody of each ESR1, ESR2, or
PGR for 1 h (
PBST and PBS, for PGR detection tissues were incubated with second antibodies
for 1 h and washed, but for ESR1 or ESR2 this step was skipped because the first
antibodies were fluorescence conjugated. The tissues were counterstained with
YOYO-1 (Cat #: Y3601, Invitrogen, Waltham, MA) for 15 min and mounted.
Specific signaling of ESR1, ESR2, or PGR was observed under the fluorescence
microscope (Zeiss Axio Observer Z1).
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