Sections were washed in running tap

Sections were washed in running tap water for 5 min; rinsed in
95% alcohol (10 dips); counterstained in eosin Y (C.I. #45380,
National Aniline and Chemical Company, Buffalo, NY) solution
for 30 sec to 1 min; dehydrated through 95% alcohol and 2
changes of absolute alcohol, 2 min each; cleared in Histo-Clear
(2X each for 5 min); and coverslipped using Permount (VWR,
Radnor, PA), a xylene based mounting medium.
Immunohistochemistry
For fluorescence immunohistochemistry, thin tissue sections
(5μm) from healthy gorgonian coral were deparaffinized and
hydrated through a descending series of ethanols (explained
previously). Sections were permeabilized in 0.05% Triton
X-100 (Sigma Aldrich, St. Louis, MO) for 15 min at room temperature.
Then, tissue sections were rinsed in filtered seawater
and incubated for 1 hour at room temperature in a blocking
solution (0.05% Triton X-100; 3% of normal goat serum;
Jackson ImmunoResearch, West Grove, PA) in filtered seawater
to prevent non-specific binding. After blocking, the tissue
sections were rinsed with filtered seawater for 5 min and
then incubated with a rabbit polyclonal primary antibody
(1/500 diluted in blocking solution) against Aspergillus (Abcam,
Cambridge, MA) at 4oC for 24 hours. Next, tissue sections were
rinsed with filtered seawater for 5 min and immediately after,
to prevent any endogenous fluorescence, tissue sections were
blocked with 0.1% sodium borohydrate for 30 mins. Tissue
sections were rinsed again with filtered seawater for 5 min
and incubated for 2 hours at room temperature with Alexa
Fluor 546® (Invitrogen, Eugene, OR) goat anti-rabbit secondary
antibody (1/200 diluted in blocking solution) (Life Technology,
Carlsbad, CA). Finally, tissue sections were washed twice (30
s each) in filtered seawater and then labeled with Alexa Fluor
488 Phalloidin (Life Technology, Carlsbad, CA) for 5 min, rinsed
again in filtered seawater (2X each 30 s), and nuclei were
stained with TO-PRO-3® (1/500 diluted in filtered seawater;
Life Technology, Carlsbad, CA). Control slides were prepared
precisely as described but utilizing the primary antibody
only. Additional controls utilizing secondary antibodies only,
were also performed. No immunohistochemical labeling was
performed on A. cervicornis.
Image acquisition
Tissue sections were observed using appropriate filter sets for
Alexa 546, phalloidin 488, and TO-PRO-3® (642/661nm) in a
Nikon Eclipse 800 (E800) optical microscope equipped with
the modular Nikon C1 Plus laser scanning confocal system
that is fully controlled by Nikon Elements AR (NISO) software.
The E800 is equipped with differential interference contrast
(DIC) and 10X Plan Fluor (N.A.=0.30), 20X Plan Fluor (N.A.=0.45),
40X Plan Fluor (N.A.=0.65), 60X S Fluor with correction collar
ring (N.A.=0.85), and 100X Plan Apo Oil immersed (N.A.=1.40)
objectives. Images were acquired with a QImaging Retiga Exi
camera (QImaging, Canada) with color filter. Image calibration
and measurements were performed using Nikon NIS-Elements.
Adobe Photoshop CS4 was used for figure plate preparation.
Results
The methods described in this report provided high quality
longitudinal and cross tissue slides from corals (e.g., polyps).
For instance, a longitudinal section of a sea fan polyp retracted
into its calyx, with 4 extended tentacles surrounding the
mouth is shown in Figure 2a. Although an artifact is observed
in the separation of the basal tissue from the axial skeleton,
this artifact is also frequently observed on histological preparations
of octocoral using conventional techniques [16,17].
In fact, the microwave technique seems to minimize this effect.
A cross sectional view of a polyp showing the mouth surrounded
by 8 hollow radial chambers lined by gastrodermal
tissue is also observed (Figure 2b). Many minute mesoglea
openings surrounding the polyp are shown, some of which
have spindle-like shapes [18]. These openings are lacunae
remaining after sclerites were decalcified. In addition, another
cross sectional view of a polyp shows a mouth surrounded by
8 tentacles filled with gastrodermal tissue (Figure 2c), each of
which are separated by a septum also lined with gastrodermal
tissue (Figure 2d).

Hasil (Bahasa Indonesia) 2:

Bagian dicuci dalam menjalankan air keran selama 5 menit; dibilas di
95% alkohol (10 dips); counterstained di Eosin Y (CI # 45380,
anilina Nasional dan Chemical Company, Buffalo, NY) solusi
untuk 30 detik untuk 1 menit; dehidrasi melalui 95% alkohol dan 2
perubahan alkohol absolut, 2 menit setiap; dibersihkan di Histo-jelas
(2X masing-masing untuk 5 menit); dan coverslipped menggunakan Permount (VWR,
Radnor, PA), sebuah xylene medium mounting. berdasarkan
Imunohistokimia
Untuk fluoresensi imunohistokimia, bagian jaringan tipis
(5μm) dari karang gorgonian sehat deparaffinized dan
terhidrasi melalui serangkaian turun dari ethanols (dijelaskan
sebelumnya). Bagian yang permeabilized di 0,05% Triton
X-100 (Sigma Aldrich, St. Louis, MO) selama 15 menit pada suhu kamar.
Kemudian, bagian jaringan dibilas dalam air laut disaring
dan diinkubasi selama 1 jam pada suhu kamar dalam blocking
solusi (0,05 % Triton X-100, 3% dari serum kambing normal,
Jackson ImmunoResearch, West Grove, PA) dalam air laut disaring
untuk mencegah non-spesifik mengikat. Setelah memblokir, jaringan
bagian dibilas dengan air laut disaring selama 5 menit dan
kemudian diinkubasi dengan kelinci poliklonal antibodi primer
(1/500 diencerkan dalam menghalangi solusi) terhadap Aspergillus (Abcam,
Cambridge, MA) pada suhu 4oC selama 24 jam. Berikutnya, bagian jaringan yang
dibilas dengan air laut disaring selama 5 menit dan segera setelah itu,
untuk mencegah fluoresensi endogen, bagian jaringan yang
diblokir dengan 0,1% sodium borohydrate selama 30 menit. Tissue
bagian dibilas lagi dengan air laut disaring selama 5 menit
dan diinkubasi selama 2 jam pada suhu kamar dengan Alexa
Fluor 546® (Invitrogen, Eugene, OR) kambing anti-kelinci sekunder
antibodi (1/200 diencerkan dalam menghalangi solusi) (Hidup Teknologi,
Carlsbad, CA). Akhirnya, bagian jaringan dicuci dua kali (30
s masing-masing) di disaring air laut dan kemudian diberi label dengan Alexa Fluor
488 Phalloidin (Hidup Teknologi, Carlsbad, CA) selama 5 menit, dibilas
lagi disaring air laut (2X setiap 30 s), dan inti yang
diwarnai dengan TO-PRO-3® (1/500 diencerkan dalam air laut disaring;
Hidup Teknologi, Carlsbad, CA). Slide kontrol disiapkan
tepat seperti yang dijelaskan tapi memanfaatkan antibodi primer
saja. Kontrol tambahan memanfaatkan antibodi sekunder saja,
juga dilakukan. Tidak ada pelabelan imunohistokimia yang
dilakukan pada A. cervicornis.
Gambar akuisisi
bagian jaringan yang diamati menggunakan set filter yang sesuai untuk
Alexa 546, Phalloidin 488, dan TO-PRO-3® (642 / 661nm) di
Nikon Eclipse 800 (E800) mikroskop optik dilengkapi dengan
modular Nikon C1 Ditambah sistem laser scanning confocal
yang sepenuhnya dikendalikan oleh Nikon Elements AR (NISO) perangkat lunak.
E800 ini dilengkapi dengan diferensial gangguan kontras
(DIC) dan 10x Rencana Fluor (NA = 0,30), 20X Rencana Fluor (NA = 0,45),
40X Rencana Fluor (NA = 0,65), 60X S Fluor dengan koreksi kerah
cincin (NA = 0,85), dan 100X Rencana Apo Minyak tenggelam (NA = 1,40)
tujuan. Gambar diperoleh dengan QImaging Retiga Exi
kamera (QImaging, Kanada) dengan filter warna. Kalibrasi gambar
dan pengukuran dilakukan dengan menggunakan Nikon NIS-Elements.
Adobe Photoshop CS4 digunakan untuk persiapan piring angka.
Hasil
Metode yang dijelaskan dalam laporan ini memberikan kualitas tinggi
slide jaringan longitudinal dan lintas dari karang (misalnya, polip).
Misalnya, longitudinal bagian dari polip penggemar laut ditarik
ke kelopak, dengan 4 tentakel diperpanjang sekitar
mulut ditunjukkan pada Gambar 2a. Meskipun artefak diamati
dalam pemisahan jaringan basal dari kerangka aksial,
artefak ini juga sering diamati pada persiapan histologis
dari octocoral menggunakan teknik konvensional [16,17].
Bahkan, teknik microwave tampaknya untuk meminimalkan efek ini.
Sebuah penampang melintang dari polip menunjukkan mulut dikelilingi
oleh 8 ruang radial berongga dilapisi oleh gastrodermal
jaringan juga diamati (Gambar 2b). Banyak menit mesoglea
bukaan sekitar polip ditunjukkan, beberapa di antaranya
memiliki spindle-seperti bentuk [18]. Lubang ini adalah kekosongan
yang tersisa setelah sclerites yang dekalsifikasi. Selain itu, yang lain
melihat cross sectional dari polip menunjukkan mulut dikelilingi oleh
tentakel 8 diisi dengan jaringan gastrodermal (Gambar 2c), masing-masing
yang dipisahkan oleh septum juga dilapisi dengan gastrodermal
jaringan (Gambar 2d).