Pap pen

Manufactured by Thermo Fisher Scientific

Sourced in Denmark

The PAP pen is a laboratory equipment designed to apply liquid wax or hydrophobic barrier on microscope slides. It creates a hydrophobic boundary to confine samples or reagents within a defined area on the slide surface.

Automatically generated - may contain errors

Lab products found in correlation

Zen 2010, by Zeiss (2 mentions) Oct compound, by Thermo Fisher Scientific (1 mentions) Gl7 fitc, by BioLegend (1 mentions) Optimal cutting temperature compound, by Thermo Fisher Scientific (1 mentions) Prolong diamond, by Thermo Fisher Scientific (1 mentions) Vectashield antifade mounting media, by Vector Laboratories (1 mentions) Lsm 780 microscope, by Zeiss (1 mentions) Axiocam mrm camera, by Zeiss (1 mentions) Zen imaging software, by Zeiss (1 mentions) Mab414, by Fortrea (1 mentions) Alexa fluor 594 secondary antibody, by Jackson ImmunoResearch (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)

5 protocols using pap pen

Immunohistochemistry of Salmonella Infection

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A full list of antibodies used for immunohistochemistry can be found
in the Key Resources table. Spleens were harvested, frozen in OCT compound
(Fisher Scientific), and frozen sections 8 m in thickness were placed on
SuperFrost Plus cryosection slides (Fisher Scientific). Sections were fixed
in ice-cold acetone at −20 °C for 10 minutes and then allowed
to dry. A boundary was drawn around tissue sections using a pap pen (Fisher
Scientific). Sections were washed with PBS and then blocked with staining
buffer (PBS with 3% bovine serum albumin, ± 1% saponin, 5 % normal
mouse serum) for 30 min at room temperature. After blocking, sections were
stained with the primary anti-Salmonella antibody in
staining buffer for 4 hours, or with primary antibodies against surface
antigens for 1.5 hours, at room temperature. Sections were washed and then
stained for 2 hours at room temperature with fluorescent conjugated
secondary antibodies. Slides were washed in PBS and then mounted using
ProLong Diamond (Life Technologies). Images were acquired on a Zeiss LSM 700
or 880 confocal microscope with the ZEN 2010 software (Zeiss) and processed
using FIJI software.

Pham T.H., Brewer S.M., Thurston T., Massis L.M., Honeycutt J., Lugo K., Jacobson A.R., Vilches-Moure J.G., Hamblin M., Helaine S, & Monack D.M. (2019). Salmonella-driven polarization of granuloma macrophages antagonizes TNF-mediated pathogen restriction during persistent infection. Cell host & microbe, 27(1), 54-67.e5.

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Splenocyte Immunofluorescence Staining Protocol

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Spleens were collected and immediately frozen in OCT buffer (Fisher Scientific) in a plastic cassette using liquid nitrogen and stored at −80 °C. Blocks were sliced into 10 μm sections using a cryostat, placed onto slides, and left to dry for 30 min. A hydrophobic barrier was drawn around sections using a PAP Pen (Fisher Scientific). Sections were fixed in 4% PFA for 15 min, washed, and then blocked in 5% rat serum + 0.5% IGEPAL in PBS for 1 h at room temperature. A primary antibody cocktail including 1:50 dilution GL7-FITC (Biolegend, clone GL7, Cat. No. 144603), 1:100 dilution CD21/35-PE (Biolegend, clone 7E9, Cat. No. 123409), and 1:100 dilution IgD-AF647 (Biolegend, clone 11–26 c.2a, Cat. No. 405707) in blocking solution was added to sections overnight at room temperature. Slides were then washed and one drop of Fluoroshield mounting medium with DAPI stain (Abcam) was added and a coverslip was placed on top.

Crouse B., Robinson C., Huseby Kelcher A., Laudenbach M., Abrahante J.E, & Pravetoni M. (2020). Mechanisms of interleukin 4 mediated increase in efficacy of vaccines against opioid use disorders. NPJ Vaccines, 5, 99.

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Immunofluorescence Staining of Frozen Spleen Sections

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Spleens were harvested and frozen in optimal cutting temperature compound (Thermo Fisher Scientific), and frozen sections (8 μm in thickness) were placed on SuperFrost Plus cryosection slides (Thermo Fisher Scientific). Sections were fixed in ice-cold acetone at −20°C for 10 min and then allowed to dry. A boundary was drawn around tissue sections using a pap pen (Thermo Fisher Scientific). Sections were washed with PBS and then blocked with a staining buffer (PBS with 3% bovine serum albumin and 5% normal mouse serum) for 30 min at room temperature. After blocking, sections were stained with the primary antibodies in a staining buffer for 2 hours at room temperature. Sections were washed and then stained for 2 hours at room temperature with fluorescent-conjugated secondary antibodies. A complete list of antibodies used in this study is provided in table S1. Slides were washed in PBS and then mounted using ProLong Diamond (Life Technologies). Images were acquired on a Zeiss LSM 700 or 880 confocal microscope with the ZEN 2010 software (Zeiss) and processed using Fiji software.

Pham T.H., Xue Y., Brewer S.M., Bernstein K.E., Quake S.R, & Monack D.M. (2023). Single-cell profiling identifies ACE+ granuloma macrophages as a nonpermissive niche for intracellular bacteria during persistent Salmonella infection. Science Advances, 9(1), eadd4333.

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Immunolocalization of Nuclear Pore Complex

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Worms were transferred to a poly-l-lysine microscopy slide (VWR, Denmark), the cuticle was punctured with a sharp needle to allow for better exposure of the germline. A coverslip was gently placed on top and freeze-cracking was performed by incubation at −80 °C followed by removal of the coverslip from the frozen slides with a scalpel. The slides were incubated 20 min in ice cold methanol, washed in PBS, and then blocked 2 h in 2% (w/v) milk-PBS. Worms were encircled with a PAP pen (ThermoFischer, Denmark) before incubation with primary antibody against nuclear pore complex (mab414, Covance) 1:1000 dilution in 2% milk-PBS overnight. After incubation, the slides were washed 2 times in PBS and incubated 2 h with goat anti-mouse alexa546 conjugated antibody (ThermoFischer, Denmark) in 2% milk-PBS. The slides were washed 3 times in PBS, fixed 10 min in 2% PFA, and mounted with Vectashield antifade mounting media (Vector Laboratories). Confocal microscopy for co-localization of DLC-1::GFP and nuclear membrane was performed using a Zeiss LSM 780 microscope equipped with a Zeiss AxioCam MRm camera. The ZEN Imaging Software (Zeiss) was used to process the pictures.

Harders R.H., Morthorst T.H., Lande A.D., Hesselager M.O., Mandrup O.A., Bendixen E., Stensballe A, & Olsen A. (2018). Dynein links engulfment and execution of apoptosis via CED-4/Apaf1 in C. elegans. Cell Death & Disease, 9(10), 1012.

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Immunohistochemical Staining of p-Tau and DARPP-32

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Slides stored at −80 °C were warmed to room temperature for 5 min and the tissue sections outlined with a PAP pen (Thermo Fisher) and gently rehydrated with 1× PBS. Sections were then incubated with permeabilization buffer (0.25% Triton X-100 in PBS), rinsed, incubated in blocking serum (5% normal donkey serum, 1% BSA, in 1× PBS) for 2 h, then incubated with rabbit anti-p-tau-Thr205 (#49561, Cell Signaling Technology; 1:100) and mouse anti-DARPP-32 conjugated to Alexa Fluor^® (sc-271111 AF647; Santa Cruz; 1:500) antibodies overnight at 4 °C. Following incubation in primary antibodies, slides were rinsed 3 × 10 min in Tris-buffered saline/Tween-20 (TBS-T; containing 0.05% Tween 20) and incubated in donkey anti-rabbit Alexa Fluor 594 secondary antibody (#711585152, Jackson ImmunoResearch; 1:400) for 1 h. Slides were rinsed 3 × 10 min in TBS-T, incubated with Hoechst 33342 (#H3570, Invitrogen, 1:20000) for 10 min to identify nuclei, followed by a final 3 × 5 min rinse in TBS-T. Slides were mounted in ProLong Gold Antifade reagent (Invitrogen, P36930).

Lark A.R., Silva L.K., Nass S.R., Marone M.G., Ohene-Nyako M., Ihrig T.M., Marks W.D., Yarotskyy V., McQuiston A.R., Knapp P.E, & Hauser K.F. (2022). Progressive degeneration and adaptive excitability in dopamine D1 and D2 receptor-expressing striatal neurons exposed to HIV-1 Tat and morphine. Cellular and molecular neurobiology, 43(3), 1105-1127.

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